Substituted indolyl alkyl amino derivatives as novel inhibitors of histone deacetylase

ABSTRACT

This invention comprises the novel compounds of formula (I) 
                         
wherein R 1 , R 2 , R 3 , X and Y have defined meanings, having histone deacetylase inhibiting enzymatic activity; their preparation, compositions containing them and their use as a medicine.

This application is a Divisional of U.S. application Ser. No.14/244,046, filed Apr. 3, 2014, pending, Continuation of U.S.application Ser. No. 13/962,523, filed Aug. 8, 2013, abandoned, which isa Divisional of U.S. application Ser. No. 13/027,793, filed Feb. 15,2011, now U.S. Pat. No. 8,524,728, which is a Continuation of U.S.application Ser. No. 11/572,563, filed Jan. 23, 2007, now U.S. Pat. No.8,193,205, which is a U.S. National Stage Filing under 35 U.S.C. §371 ofPCT/EP2005/053612, filed Jul. 25, 2005. PCT/EP2005/053612 claimspriority to U.S. Provisional Patent Application No. 60/592,182, filedJul. 29, 2004 and EPO Patent Application No. 04077172.7, filed Jul. 28,2004. The entire disclosures of each of these patent applications arehereby incorporated in their entirety.

This invention concerns compounds having histone deacetylase (HDAC)inhibiting enzymatic activity. It further relates to processes for theirpreparation, to compositions comprising them, as well as their use, bothin vitro and in vivo, to inhibit HDAC and as a medicine, for instance asa medicine to inhibit proliferative conditions, such as cancer andpsoriasis.

Nuclear histones are known as integral and dynamic components of themachinery responsible for regulating gene transcription and otherDNA-templated processes such as replication, repair, recombination, andchromosome segregation. They are the subject of post-translationalmodifications including acetylation, phosphorylation, methylation,ubiquitination, and ADP-ribosylation.

Histone deacetylase(s), herein referred to as “HDACs”, are enzymes thatcatalyze the removal of the acetyl modification on lysine residues ofproteins, including the core nucleosomal histones H2A, H2B, H3 and H4.Together with histone acetyltransferase(s), herein referred to as“HATs”, HDACs regulate the level of acetylation of the histones. Thebalance of acetylation of nucleosomal histones plays an important rolein transcription of many genes. Hypoacetylation of histones isassociated with condensed chromatin structure resulting in therepression of gene transcription, whereas acetylated histones areassociated with a more open chromatin structure and activation oftranscription.

Eleven structurally related HDACs have been described and fall into twoclasses. Class I HDACs consist of HDAC 1, 2, 3, 8 and 11 whereas classII HDACs consist of HDAC 4, 5, 6, 7, 9 and 10. Members of a third classof HDACs are structurally unrelated to the class I and class II HDACs.Class I/II HDACs operate by zinc-dependent mechanisms, whereas class IIIHDACs are NAD-dependent.

In addition to histones, other proteins have also been the substrate foracetylation, in particular transcriptionfactors such as p53, GATA-1 andE2F; nuclear receptors such as the glucocorticoid receptor, the thyroidreceptors, the estrogen receptors; and cell-cycle regulating proteinssuch as pRb. Acetylation of proteins has been linked with proteinstabilization, such as p53 stabilization, recruitment of cofactors andincreased DNA binding. p53 is a tumour suppressor that can induce cellcycle arrest or apoptosis in response to a variety of stress signals,such as DNA damage. The main target for p53-induced cell cycle arrestseems to be the p21 gene. Next to its activation by p53, p21 has beenidentified by virtue of its association with cyclin/cyclin-dependentkinase complexes resulting in cell cycle arrest at both G1 and G2phases, its up-regulation during senescence, and its interaction withthe proliferating cell nuclear antigen.

The study of inhibitors of HDACs indicates that they play an importantrole in cell cycle arrest, cellular differentiation, apoptosis andreversal of transformed phenotypes.

The inhibitor Trichostatin A (TSA), for example, causes cell cyclearrest at both G1 and G2 phases, reverts the transformed phenotype ofdifferent cell lines, and induces differentiation of Friend leukemiacells and others. TSA (and suberoylanilide hydroxamic acid SAHA) havebeen reported to inhibit cell growth, induce terminal differentiation,and prevent the formation of tumours in mice (Finnin et al., Nature,401: 188-193, 1999).

Trichostatin A has also been reported to be useful in the treatment offibrosis, e.g. liver fibrosis and liver chirrhosis. (Geerts et al.,European Patent Application EP 0 827 742, published 11 Mar. 1998).

The pharmacophore for HDAC inhibitors consists of a metal-bindingdomain, which interacts with the zinc-containing active site of HDACs, alinker domain, and a surface recognition domain or capping region, whichinteracts with residues on the rim of the active site.

Inhibitors of HDACs have also been reported to induce p21 geneexpression. The transcriptional activation of the p21 gene by theseinhibitors is promoted by chromatin remodelling, following acetylationof histones H3 and H4 in the p21 promotor region. This activation of p21occurs in a p53-independent fashion and thus HDAC inhibitors areoperative in cells with mutated p53 genes, a hallmark of numeroustumours.

In addition HDAC inhibitors can have indirect activities such asaugmentation of the host immune respons and inhibition of tumorangiogenesis and thus can suppress the growth of primary tumors andimpede metastasis (Mai et al., Medicinal Research Reviews, 25: 261-309).

In view of the above, HDAC inhibitors can have great potential in thetreatment of cell proliferative diseases or conditions, includingtumours with mutated p53 genes.

Patent application EP1472216 published on Aug. 14, 2003 disclosesbicyclic hydroxamates as inhibitors of histone deacetylase.

Patent applications EP1485099, EP1485348, EP1485353, EP1485354,EP1485364, EP1485365, EP1485370, EP1485378 published on 18 Sep. 2003,amongst others, disclose substituted piperazinylpyrimidinylhydroxamicacids as inhibitors of histone deacetylase furthermore EP1485365discloses R306465.

Patent application EP1492534 published on 9 Oct. 2003, disclosescarbamic acid compounds comprising a piperazine linkage, as HDACinhibitors.

Patent application EP1495002 published on 23 Oct. 2003, disclosesubstituted piperazinyl phenyl benzamide compounds, as histonedeacetylase inhibitors.

Patent application WO04/009536 published on 29 Jan. 2004, disclosesderivatives containing an alkyl linker between the aryl group and thehydroxamate, as histone deacetylase inhibitors.

Patent application EP1525199 published on 12 Feb. 2004, discloses(hetero)arylalkenyl substituted bicyclic hydroxamates, as histonedeacetylase inhibitors.

Patent application WO04/063146 published on 29 Jul. 2004, disclosesderivatives of N-hydroxy-benzamide derivatives with anti-inflammatoryand antitumour activity.

Patent application WO04/063169 published on 29 Jul. 2004, disclosessubstituted aryl hydroxamate derivatives as histone deacetylaseinhibitors.

Patent application WO04/072047 published on 26 Aug. 2004, disclosesindoles, benzimidazoles and naphhimidazoles as histone deacetylaseinhibitors.

Patent application WO04/082638 published on 30 Sep. 2004, discloseshydroxamates linked to non-aromatic heterocyclic ring systems as histonedeacetylase inhibitors.

Patent application WO04/092115 published on 28 Oct. 2004, discloseshydroxamate derivatives as histone deacetylase inhibitors.

Patent application WO05/028447 published on 31 Mar. 2005, disclosesbenzimidazoles as histone deacetylase inhibitors.

Patent applications WO05/030704 and WO05/030705 published on 7 Apr.2005, discloses benzamides as histone deacetylase inhibitors.

Patent application WO05/040101 published on 6 May 2005, disclosesacylurea connected and sulfonylurea connected hydroxamates as histonedeacetylase inhibitors.

Patent application WO05/040161 also published on 6 May 2005, disclosesbiaryl linked hydroxamates as histone deacetylase inhibitors.

The compounds of the present invention differ from the prior art instructure, in their pharmacological activity and/or pharmacologicalpotency.

The problem to be solved is to provide histone deacetylase inhibitorswith high enzymatic and cellular activity that have increasedbioavailability and/or in vivo potency.

The novel compounds of the present invention solve the above-describedproblem. The compounds of the present invention show excellent histonedeacetylase inhibiting enzymatic and cellular activity. They have a highcapacity to activate the p21 gene, both at the cellular and the in vivolevel. They have a desirable pharmacokinetic profile and low affinityfor the P450 enzymes, which reduces the risk of adverse drug-druginteraction allowing also for a wider safety margin.

Advantageous features of the present compounds are metabolic stability,solubility and/or p21 induction capacity. More in particular thecompounds of the present invention have increased half-lives in rathepatocytes, have an increased solubility/stability in aqueous solutionsand/or have enhanced in vivo p21 promoter inducing capacities.

This invention concerns compounds of formula (I)

the N-oxide forms, the pharmaceutically acceptable addition salts andthe stereo-chemically isomeric forms thereof, wherein

each n is an integer with value 0, 1 or 2 and when n is 0 then a directbond is intended;

each m is an integer with value 1 or 2;

each X is independently N or CH;

each Y is independently O, S, or NR⁴; wherein

-   each R⁴ is hydrogen, C₁₋₆alkyl, C₁₋₆alkyloxyC₁₋₆alkyl,    C₃₋₆cycloalkyl, C₃₋₆cycloalkylmethyl, phenylC₁₋₆alkyl, —C(═O)—CHR⁵R⁶    or —S(═O)₂—N(CH₃)₂; wherein-   each R⁵ and R⁶ is independently hydrogen, amino, C₁₋₆alkyl or    aminoC₁₋₆alkyl; and-   when Y is NR⁴ and R² is on the 7-position of the indolyl then R² and    R⁴ together can form the bivalent radical    —(CH₂)₂—  (a-1), or    —(CH₂)₃—  (a-2);-   R¹ is hydrogen, C₁₋₆alkyl, hydroxyC₁₋₆alkyl, C₁₋₆alkylsulfonyl,    C₁₋₆alkylcarbonyl or mono- or di(C₁₋₆alkyl)aminosulfonyl;-   R² is hydrogen, hydroxy, amino, halo, C₁₋₆alkyl, cyano, C₂₋₆alkenyl,    polyhaloC₁₋₆alkyl, nitro, phenyl, C₁₋₆alkylcarbonyl,    hydroxycarbonyl, C₁₋₆alkylcarbonylamino, C₁₋₆alkyloxy, or mono- or    di(C₁₋₆alkyl)amino;-   R³ is hydrogen, C₁₋₆alkyl, or C₁₋₆alkyloxy; and-   when R² and R³ are on adjacent carbon atoms, they can form the    bivalent radical —O—CH₂—O—.

Lines drawn into the bicyclic ring systems from substituents indicatethat the bonds may be attached to any of the suitable ring atoms of thebicyclic ring system.

The term “histone deacetylase inhibitor” or “inhibitor of histonedeacetylase” is used to identify a compound, which is capable ofinteracting with a histone deacetylase and inhibiting its activity, moreparticularly its enzymatic activity. Inhibiting histone deacetylaseenzymatic activity means reducing the ability of a histone deacetylaseto remove an acetyl group from a histone. Preferably, such inhibition isspecific, i.e. the histone deacetylase inhibitor reduces the ability ofa histone deacetylase to remove an acetyl group from a histone at aconcentration that is lower than the concentration of the inhibitor thatis required to produce some other, unrelated biological effect.

As used in the foregoing definitions and hereinafter, halo is generic tofluoro, chloro, bromo and iodo; C₁₋₂alkyl straight chain saturatedhydrocarbon radicals having 1 or 2 carbon atoms such as, e.g. methyl orethyl; C₁₋₆alkyl defines C₁₋₂alkyl and straight and branched chainsaturated hydrocarbon radicals having from 3 to 6 carbon atoms such as,e.g. propyl, butyl, 1-methylethyl, 2-methylpropyl, pentyl,2-methyl-butyl, hexyl, 2-methylpentyl and the like; andpolyhaloC₁₋₆alkyl defines C₁₋₆alkyl containing three identical ordifferent halo substituents for example trifluoromethyl; C₂₋₆alkenyldefines straight and branched chain hydrocarbon radicals containing onedouble bond and having from 2 to 6 carbon atoms such as, for example,ethenyl, 2-propenyl, 3-butenyl, 2-pentenyl, 3-pentenyl,3-methyl-2-butenyl, and the like; C₃₋₆cycloalkyl includes cyclichydrocarbon groups having from 3 to 6 carbons, such as cyclopropyl,cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, andthe like.

Pharmaceutically acceptable addition salts encompass pharmaceuticallyacceptable acid addition salts and pharmaceutically acceptable baseaddition salts. The pharmaceutically acceptable acid addition salts asmentioned hereinabove are meant to comprise the therapeutically activenon-toxic acid addition salt forms, which the compounds of formula (I)are able to form. The compounds of formula (I) which have basicproperties can be converted in their pharmaceutically acceptable acidaddition salts by treating said base form with an appropriate acid.Appropriate acids comprise, for example, inorganic acids such ashydrohalic acids, e.g. hydrochloric or hydrobromic acid; sulfuric;nitric; phosphoric and the like acids; or organic acids such as, forexample, acetic, trifluoroacetic, propanoic, hydroxyacetic, lactic,pyruvic, oxalic, malonic, succinic (i.e. butanedioic acid), maleic,fumaric, malic, tartaric, citric, methanesulfonic, ethanesulfonic,benzenesulfonic, p-toluenesulfonic, cyclamic, salicylic,p-amino-salicylic, pamoic and the like acids.

The compounds of formula (I) which have acidic properties may beconverted in their pharmaceutically acceptable base addition salts bytreating said acid form with a suitable organic or inorganic base.Appropriate base salt forms comprise, for example, the ammonium salts,the alkali and earth alkaline metal salts, e.g. the lithium, sodium,potassium, magnesium, calcium salts and the like, salts with organicbases, e.g. the benzathine, N-methyl-D-glucamine, hydrabamine salts, andsalts with amino acids such as, for example, arginine, lysine and thelike.

The term “acid or base addition salts” also comprises the hydrates andthe solvent addition forms, which the compounds of formula (I) are ableto form. Examples of such forms are e.g. hydrates, alcoholates and thelike.

The term “stereochemically isomeric forms of compounds of formula (I)”,as used herein, defines all possible compounds made up of the same atomsbonded by the same sequence of bonds but having differentthree-dimensional structures, which are not interchangeable, which thecompounds of formula (I) may possess. Unless otherwise mentioned orindicated, the chemical designation of a compound encompasses themixture of all possible stereochemically isomeric forms, which saidcompound may possess. Said mixture may contain all diastereomers and/orenantiomers of the basic molecular structure of said compound. Allstereochemically isomeric forms of the compounds of formula (I) both inpure form or in admixture with each other are intended to be embracedwithin the scope of the present invention.

The N-oxide forms of the compounds of formula (I) are meant to comprisethose compounds of formula (I) wherein one or several nitrogen atoms areoxidized to the so-called N-oxide, particularly those N-oxides whereinone or more of the piperidine-, piperazine or pyridazinyl-nitrogens areN-oxidized.

Some of the compounds of formula (I) may also exist in their tautomericforms. Such forms although not explicitly indicated in the above formulaare intended to be included within the scope of the present invention.

Whenever used hereinafter, the term “compounds of formula (I)” is meantto include also the pharmaceutically acceptable addition salts and allstereoisomeric forms.

As used herein, the terms “histone deacetylase” and “HDAC” are intendedto refer to any one of a family of enzymes that remove acetyl groupsfrom the c-amino groups of lysine residues at the N-terminus of ahistone. Unless otherwise indicated by context, the term “histone” ismeant to refer to any histone protein, including H1, H2A, H2B, H3, H4,and H5, from any species. Human HDAC proteins or gene products, include,but are not limited to, HDAC-1, HDAC-2, HDAC-3, HDAC-4, HDAC-5, HDAC-6,HDAC-7, HDAC-8, HDAC-9 HDAC-10 and HDAC-11. The histone deacetylase canalso be derived from a protozoal or fungal source.

A first group of interesting compounds consists of those compounds offormula (I) wherein one or more of the following restrictions apply:

-   a) each R⁴ is hydrogen, C₁₋₆alkyl, C₃₋₆cycloalkyl,    C₃₋₆cycloalkylmethyl, —C(═O)—CHR⁵R⁶ or —S(═O)₂—N(CH₃)₂;-   b) R¹ is hydrogen, C₁₋₆alkyl, hydroxyC₁₋₆alkyl, C₁₋₆alkylsulfonyl,    or mono- or di(C₁₋₆alkyl)aminosulfonyl; or-   c) R³ is hydrogen.

A second group of interesting compounds consists of those compounds offormula (I) wherein one or more of the following restrictions apply:

-   a) each n is an integer with value 0 or 1;-   b) each X is independently N;-   c) each R⁴ is hydrogen or C₁₋₆alkyl;-   d) R¹ is hydrogen, C₁₋₆alkyl or hydroxyC₁₋₆alkyl; or-   e) R² is hydrogen, halo, C₁₋₆alkyl or C₁₋₆alkyloxy.

A third group of interesting compounds consists of those compounds offormula (I) wherein one or more of the following restrictions apply:

-   a) each n is an integer with value 0 or 1;-   b) each R⁴ is hydrogen, C₁₋₆alkyl, C₁₋₆alkyloxyC₁₋₆alkyl,    C₃₋₆cycloalkyl or phenylC₁₋₆alkyl;-   c) R¹ is hydrogen, C₁₋₆alkyl, hydroxyC₁₋₆alkyl, C₁₋₆alkylcarbonyl or    C₁₋₆alkylsulfonyl; or-   d) R² is hydrogen, halo, C₁₋₆alkyl, cyano, nitro or C₁₋₆alkyloxy.

A fourth group of interesting compounds consists of those compounds offormula (I) wherein one or more of the following restrictions apply:

-   a) each n is an integer with value 0 or 1;-   b) each m is an integer with value 1;-   c) each R⁴ is hydrogen, C₁₋₆alkyloxyC₁₋₆alkyl, C₃₋₆cycloalkyl or    phenylC₁₋₆alkyl;-   c) R¹ is hydrogen, hydroxyC₁₋₆alkyl, C₁₋₆alkylcarbonyl or    C₁₋₆alkylsulfonyl;-   d) R² is hydrogen, halo, cyano, nitro or C₁₋₆alkyloxy; or-   e) R³ is C₁₋₆alkyloxy; or-   f) when R² and R³ are on adjacent carbon atoms, they can form the    bivalent radical —O—CH₂—O—.

A fifth group of interesting compounds consists of those compounds offormula (I) wherein one or more of the following restrictions apply:

-   a) each n is an integer with value 1;-   b) each m is an integer with value 1;-   c) each X is independently N;-   d) each Y is independently NR⁴;-   e) each R⁴ is C₁₋₆alkyl;-   f) R¹ is hydrogen;-   g) R² is hydrogen or halo; or-   h) R³ is hydrogen.

A group of preferred compounds consists of those compounds of formula(I) wherein each n is an integer with value 0 or 1; each R⁴ is hydrogen,C₁₋₆alkyl, C₁₋₆alkyloxyC₁₋₆alkyl, C₃₋₆cycloalkyl or phenylC₁₋₆alkyl; R¹is hydrogen, C₁₋₆alkyl, hydroxyC₁₋₆alkyl, C₁₋₆alkylcarbonyl orC₁₋₆alkylsulfonyl; and R² is hydrogen, halo, C₁₋₆alkyl, cyano, nitro,polyhaloC₁₋₆alkyl or C₁₋₆alkyloxy.

A group of more preferred compounds consists of those compounds offormula (I) wherein each n is an integer with value 1; each m is aninteger with value 1; each X is independently N; each Y is independentlyNR⁴; each R⁴ is C₁₋₆alkyl; R¹ is hydrogen; R² is hydrogen or halo; andR³ is hydrogen.

The most preferred compounds are compound No. 1a, compound No. 30,compound No. 39 and compound No. 50

The compounds of formula (I) and (II), their pharmaceutically acceptablesalts and N-oxides and stereochemically isomeric forms thereof may beprepared in conventional manner. The starting materials and some of theintermediates are known compounds and are commercially available or maybe prepared according to conventional reaction procedures as generallyknown in the art or as described in patent applications EP1485099,EP1485348, EP1485353, EP1485354, EP1485364, EP1485365, EP1485370, andEP1485378. Some preparation methods will be described hereinafter inmore detail. Other methods for obtaining final compounds of formula (I)are described in the examples.

a) Hydroxamic acids of formula (I) may be prepared by reacting anintermediate of formula (II), wherein Q istetrahydropyranyloxyaminocarbonyl, herein referred to as intermediatesof formula (II-a), with an appropriate acid, such as for example,trifluoro acetic acid. Said reaction is performed in an appropriatesolvent, such as, for example, methanol or dichloromethane.

b) Alternatively, hydroxamic acids of formula (I) may be prepared byreacting an intermediate of formula (II), wherein Q isC₁₋₂alkyloxycarbonyl, herein referred to as intermediates of formula(II-c), with hydroxylamine, in the presence of a base such as forexample sodium hydroxyde. Said reaction is performed in an appropriatesolvent, such as, for example, methanol.

The compounds of formula (I) may also be converted into each other viaart-known reactions or functional group transformations. A number ofsuch transformations are already described hereinabove. Other examplesare hydrolysis of carboxylic esters to the corresponding carboxylic acidor alcohol; hydrolysis of amides to the corresponding carboxylic acidsor amines; hydrolysis of nitriles to the corresponding amides; aminogroups on imidazole or phenyl may be replaced by a hydrogen by art-knowndiazotation reactions and subsequent replacement of the diazo-group byhydrogen; alcohols may be converted into esters and ethers; primaryamines may be converted into secondary or tertiary amines; double bondsmay be hydrogenated to the corresponding single bond; an iodo radical ona phenyl group may be converted in to an ester group by carbon monoxideinsertion in the presence of a suitable palladium catalyst.

The present invention also concerns intermediates of formula (II)

the N-oxide forms, the pharmaceutically acceptable addition salts andthe stereo-chemically isomeric forms thereof, wherein

-   each n is an integer with value 0, 1 or 2 and when n is 0 then a    direct bond is intended; each m is an integer with value 1 or 2;-   each X is independently N or CH;-   each Y is independently O, S, or NR⁴; wherein-   each R⁴ is hydrogen, C₁₋₆alkyl, C₁₋₆alkyloxyC₁₋₆alkyl,    C₃₋₆cycloalkyl, C₃₋₆cycloalkylmethyl, phenylC₁₋₆alkyl, —C(═O)—CHR⁵R⁶    or —S(═O)₂—N(CH₃)₂; wherein-   each R⁵ and R⁶ is independently hydrogen, amino, C₁₋₆alkyl or    aminoC₁₋₆alkyl; and-   when Y is NR⁴ and R² is on the 7-position of the indolyl then R² and    R⁴ together can form the bivalent radical    —(CH₂)₂—  (a-1), or    —(CH₂)₃—  (a-2);-   R¹ is hydrogen, C₁₋₆alkyl, hydroxyC₁₋₆alkyl, C₁₋₆alkylsulfonyl,    C₁₋₆alkylcarbonyl or mono- or di(C₁₋₆alkyl)aminosulfonyl;-   R² is hydrogen, hydroxy, amino, halo, C₁₋₆alkyl, cyano, C₂₋₆alkenyl,    polyhaloC₁₋₆alkyl, nitro, phenyl, C₁₋₆alkylcarbonyl,    hydroxycarbonyl, C₁₋₆alkylcarbonylamino, C₁₋₆ alkyloxy, or mono- or    di(C₁₋₆alkyl)amino;-   R³ is hydrogen, C₁₋₆alkyl, or C₁₋₆alkyloxy;-   when R² and R³ are on adjacent carbon atoms, they can form the    bivalent radical —O—CH₂—O—; and-   Q is C₁₋₂ alkyloxycarbonyl, hydroxycarbonyl or    tetrahydropyranyloxyaminocarbonyl.

Groups of interesting, preferred, more preferred and most preferredcompounds can be defined for the compounds of formula (II), inaccordance with the groups defined for the compounds of formula (I).

a) Intermediates of formula (II-a) may be prepared by reacting anintermediate of formula (III) with an intermediate of formula (II),wherein Q is hydroxycarbonyl, herein referred to as intermediates offormula (II-b), in the presence of appropriate reagents such asN-(ethylcarbonimidoyl)-N,N-dimethyl-1,3-propanediamine,monohydrochloride (EDC) and 1-hydroxy-1H-benzotriazole (HOBT). Thereaction may be performed in the presence of a base such astriethylamine, in a suitable solvent, such as, a mixture ofdichloromethane and tetrahydrofuran.

b) Intermediates of formula (II-a) may also be prepared by reacting anintermediate of formula (VI) with the appropriate carboxaldehyde offormula (V), wherein t is an integer with value 0 or 1, and when t is 0then a direct bond is intended, in the presence of an appropriatereagent, such as a sodium borohydride, in a suitable solvent such asdichloroethane or methanol.

c) Intermediates of formula (II-b) may be prepared by reacting anintermediate of formula (II), wherein Q is methyl- or ethyloxycarbonyl(C₁₋₂alkyl), herein referred to as intermediates of formula (II-c), withan appropriate acidic solution, e.g. hydrochloric acid, or basicsolution, e.g. hydrogen bromide or sodiumhydroxide, in a suitablesolvent e.g. an alcohol, such as ethanol or propanol.

d) The intermediates of formula (II-c) may be prepared by reacting thecarboxylic acid ethyl ester of formula (IV) with the appropriatecarboxaldehyde of formula (V), in the presence of an appropriatereagent, such as a sodium borohydride e.g. sodium tetrahydroborate, in asuitable solvent such as an alcohol e.g. methanol.

e) In an identical way, the intermediates of formula (II-c) may beprepared by reacting an intermediate of formula (XIV) with theappropriate intermediate of formula (XV), in the presence of anappropriate reagent, such as a sodium borohydride e.g. sodiumtetrahydroborate, in a suitable solvent such as an alcohol e.g.methanol.

f) Intermediates of formula (II-c) may also be prepared by reacting anintermediate of formula (X) with an intermediate of formula (XI) whereinW is an appropriate leaving group such as, for example, halo, e.g.fluoro, chloro, bromo or iodo, or a sulfonyloxy radical such asmethylsulfonyloxy,

4-methylphenylsulfonyloxy and the like. The reaction can be performed ina reaction-inert solvent such as, for example, an alcohol, e.g.methanol, ethanol, 2-methoxy-ethanol, propanol, butanol and the like; anether, e.g. 4,4-dioxane, 1,1′-oxybispropane and the like; a ketone, e.g.4-methyl-2-pentanone; or

N,N-dimethylformamide, nitrobenzene, acetonitrile and the like. Theaddition of an appropriate base such as, for example, an alkali or earthalkaline metal carbonate or hydrogen carbonate, e.g. triethylamine orsodium carbonate, may be utilized to pick up the acid which is liberatedduring the course of the reaction. A small amount of an appropriatemetal iodide, e.g., sodium or potassium iodide may be added to promotethe reaction. Stirring may enhance the rate of the reaction. Thereaction may conveniently be carried out at a temperature rangingbetween room temperature and the reflux temperature of the reactionmixture and, if desired, the reaction may be carried out at an increasedpressure.

g) In an identical way, intermediates of formula (II-c) may be preparedby reacting an intermediate of formula (XII) with an intermediate offormula (XIII), wherein W is an appropriate leaving group as describedabove.

The intermediates of formula (VI) may be prepared by reacting theintermediate of formula (VII) with piperidine in a suitable solvent e.g.dichloromethane.

The intermediates of formula (VII) may be prepared by reacting anintermediate of formula (VIII) with an intermediate of formula (III), inthe presence of appropriate reagents such asN-(ethylcarbonimidoyl)-N,N-dimethyl-1,3-propanediamine,monohydrochloride (EDC) and 1-hydroxy-1H-benzotriazole (HOBT). Thereaction may be performed in the presence of a base such astriethylamine, in a suitable solvent, such as, a mixture ofdichloromethane and tetrahydrofuran.

Intermediates of formula (VIII) may be prepared by reacting anintermediate of formula (IX) with an intermediate of formula (IV),wherein R¹ is hydrogen, herein referred to as intermediates of formula(IV-a), in the presence of sodium hydroxide, in a suitable solvent, suchas tetrahydrofuran, followed by neutralization with hydrochloric acidand addition of sodium carbonate.

The compounds of formula (I) and some of the intermediates may have atleast one stereogenic centre in their structure. This stereogenic centremay be present in an R or an S configuration.

The compounds of formula (I) as prepared in the hereinabove describedprocesses are generally racemic mixtures of enantiomers, which can beseparated from one another following art-known resolution procedures.The racemic compounds of formula (I) may be converted into thecorresponding diastereomeric salt forms by reaction with a suitablechiral acid. Said diastereomeric salt forms are subsequently separated,for example, by selective or fractional crystallization and theenantiomers are liberated there from by alkali. An alternative manner ofseparating the enantiomeric forms of the compounds of formula (I)involves liquid chromatography using a chiral stationary phase. Saidpure stereochemically isomeric forms may also be derived from thecorresponding pure stereochemically isomeric forms of the appropriatestarting materials, provided that the reaction occursstereospecifically. Preferably if a specific stereoisomer is desired,said compound would be synthesized by stereospecific methods ofpreparation. These methods will advantageously employ enantiomericallypure starting materials.

The compounds of formula (I), the pharmaceutically acceptable acidaddition salts and stereoisomeric forms thereof have valuablepharmacological properties in that they have a histone deacetylase(HDAC) inhibitory effect.

This invention provides a method for inhibiting the abnormal growth ofcells, including transformed cells, by administering an effective amountof a compound of the invention. Abnormal growth of cells refers to cellgrowth independent of normal regulatory mechanisms (e.g. loss of contactinhibition). This includes the inhibition of tumour growth both directlyby causing growth arrest, terminal differentiation and/or apoptosis ofcancer cells, and indirectly, by inhibiting neovascularization oftumours.

This invention also provides a method for inhibiting tumour growth byadministering an effective amount of a compound of the presentinvention, to a subject, e.g. a mammal (and more particularly a human)in need of such treatment. In particular, this invention provides amethod for inhibiting the growth of tumours by the administration of aneffective amount of the compounds of the present invention. Examples oftumours which may be inhibited, but are not limited to, lung cancer(e.g. adenocarcinoma and including non-small cell lung cancer),pancreatic cancers (e.g. pancreatic carcinoma such as, for exampleexocrine pancreatic carcinoma), colon cancers (e.g. colorectalcarcinomas, such as, for example, colon adenocarcinoma and colonadenoma), prostate cancer including the advanced disease, hematopoietictumours of lymphoid lineage (e.g. acute lymphocytic leukemia, B-celllymphoma, Burkitt's lymphoma), myeloid leukemias (for example, acutemyelogenous leukemia (AML)), thyroid follicular cancer, myelodysplasticsyndrome (MDS), tumours of mesenchymal origin (e.g. fibrosarcomas andrhabdomyosarcomas), melanomas, teratocarcinomas, neuroblastomas,gliomas, benign tumour of the skin (e.g. keratoacanthomas), breastcarcinoma (e.g. advanced breast cancer), kidney carcinoma, ovarycarcinoma, bladder carcinoma and epidermal carcinoma.

The compound according to the invention may be used for othertherapeutic purposes, for example:

-   -   a) the sensitisation of tumours to radiotherapy by administering        the compound according to the invention before, during or after        irradiation of the tumour for treating cancer;    -   b) treating arthropathies and osteopathological conditions such        as rheumatoid arthritis, osteoarthritis, juvenile arthritis,        gout, polyarthritis, psoriatic arthritis, ankylosing spondylitis        and systemic lupus erythematosus;    -   c) inhibiting smooth muscle cell proliferation including        vascular proliferative disorders, atherosclerosis and        restenosis;    -   d) treating inflammatory conditions and dermal conditions such        as ulcerative colitis, Crohn's disease, allergic rhinitis, graft        vs. host disease, conjunctivitis, asthma, ARDS, Behcets disease,        transplant rejection, uticaria, allergic dermatitis, alopecia        areata, scleroderma, exanthema, eczema, dermatomyositis, acne,        diabetes, systemic lupus erythematosis, Kawasaki's disease,        multiple sclerosis, emphysema, cystic fibrosis and chronic        bronchitis;    -   e) treating endometriosis, uterine fibroids, dysfunctional        uterine bleeding and endometrial hyperplasia;    -   f) treating ocular vascularisation including vasculopathy        affecting retinal and choroidal vessels;    -   g) treating a cardiac dysfunction;    -   h) inhibiting immunosuppressive conditions such as the treatment        of HIV infections;    -   i) treating renal dysfunction;    -   j) suppressing endocrine disorders;    -   k) inhibiting dysfunction of gluconeogenesis;    -   l) treating a neuropathology for example Parkinson's disease or        a neuropathology that results in a cognitive disorder, for        example, Alzheimer's disease or polyglutamine related neuronal        diseases;    -   m) treating psychiatric disorders for example schizophrenia,        bipolar disorder, depression, anxiety and psychosis;    -   n) inhibiting a neuromuscular pathology, for example,        amylotrophic lateral sclerosis;    -   o) treating spinal muscular atrophy;    -   p) treating other pathologic conditions amenable to treatment by        potentiating expression of a gene;    -   q) enhancing gene therapy;    -   r) inhibiting adipogenesis;    -   s) treating parasitosis such as malaria.

Hence, the present invention discloses the compounds of formula (I) foruse as a medicine as well as the use of these compounds of formula (I)for the manufacture of a medicament for treating one or more of theabove mentioned conditions.

The compounds of formula (I), the pharmaceutically acceptable acidaddition salts and stereoisomeric forms thereof can have valuablediagnostic properties in that they can be used for detecting oridentifying a HDAC in a biological sample comprising detecting ormeasuring the formation of a complex between a labelled compound and aHDAC.

The detecting or identifying methods can use compounds that are labelledwith labelling agents such as radioisotopes, enzymes, fluorescentsubstances, luminous substances, etc. Examples of the radioisotopesinclude ¹²⁵I, ¹³¹I, ³H and ¹⁴C. Enzymes are usually made detectable byconjugation of an appropriate substrate which, in turn catalyses adetectable reaction. Examples thereof include, for example,beta-galactosidase, beta-glucosidase, alkaline phosphatase, peroxidaseand malate dehydrogenase, preferably horseradish peroxidase. Theluminous substances include, for example, luminol, luminol derivatives,luciferin, aequorin and luciferase.

Biological samples can be defined as body tissue or body fluids.Examples of body fluids are cerebrospinal fluid, blood, plasma, serum,urine, sputum, saliva and the like.

In view of their useful pharmacological properties, the subjectcompounds may be formulated into various pharmaceutical forms foradministration purposes.

To prepare the pharmaceutical compositions of this invention, aneffective amount of a particular compound, in base or acid addition saltform, as the active ingredient is combined in intimate admixture with apharmaceutically acceptable carrier, which carrier may take a widevariety of forms depending on the form of preparation desired foradministration. These pharmaceutical compositions are desirably inunitary dosage form suitable, preferably, for administration orally,rectally, percutaneously, or by parenteral injection. For example, inpreparing the compositions in oral dosage form, any of the usualpharmaceutical media may be employed, such as, for example, water,glycols, oils, alcohols and the like in the case of oral liquidpreparations such as suspensions, syrups, elixirs and solutions; orsolid carriers such as starches, sugars, kaolin, lubricants, binders,disintegrating agents and the like in the case of powders, pills,capsules and tablets.

Because of their ease in administration, tablets and capsules representthe most advantageous oral dosage unit form, in which case solidpharmaceutical carriers are obviously employed. For parenteralcompositions, the carrier will usually comprise sterile water, at leastin large part, though other ingredients, to aid solubility for example,may be included. Injectable solutions, for example, may be prepared inwhich the carrier comprises saline solution, glucose solution or amixture of saline and glucose solution. Injectable suspensions may alsobe prepared in which case appropriate liquid carriers, suspending agentsand the like may be employed. In the compositions suitable forpercutaneous administration, the carrier optionally comprises apenetration enhancing agent and/or a suitable wetting agent, optionallycombined with suitable additives of any nature in minor proportions,which additives do not cause a significant deleterious effect to theskin. Said additives may facilitate the administration to the skinand/or may be helpful for preparing the desired compositions. Thesecompositions may be administered in various ways, e.g., as a transdermalpatch, as a spot-on or as an ointment.

It is especially advantageous to formulate the aforementionedpharmaceutical compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used in thespecification and claims herein refers to physically discrete unitssuitable as unitary dosages, each unit containing a predeterminedquantity of active ingredient, calculated to produce the desiredtherapeutic effect, in association with the required pharmaceuticalcarrier. Examples of such dosage unit forms are tablets (includingscored or coated tablets), capsules, pills, powder packets, wafers,injectable solutions or suspensions, teaspoonfuls, tablespoonfuls andthe like, and segregated multiples thereof.

Those skilled in the art could easily determine the effective amountfrom the test results presented hereinafter. In general it iscontemplated that a therapeutically effective amount would be from 0.005mg/kg to 100 mg/kg body weight, and in particular from 0.005 mg/kg to 10mg/kg body weight. It may be appropriate to administer the required doseas two, three, four or more sub-doses at appropriate intervalsthroughout the day. Said sub-doses may be formulated as unit dosageforms, for example, containing 0.5 to 500 mg, and in particular 10 mg to500 mg of active ingredient per unit dosage form.

As another aspect of the present invention a combination of aHDAC-inhibitor with another anticancer agent is envisaged, especiallyfor use as a medicine, more specifically in the treatment of cancer orrelated diseases.

For the treatment of the above conditions, the compounds of theinvention may be advantageously employed in combination with one or moreother medicinal agents, more particularly, with other anti-canceragents. Examples of anti-cancer agents are:

-   -   platinum coordination compounds for example cisplatin,        carboplatin or oxalyplatin;    -   taxane compounds for example paclitaxel or docetaxel;    -   topoisomerase I inhibitors such as camptothecin compounds for        example irinotecan or topotecan;    -   topoisomerase II inhibitors such as anti-tumour podophyllotoxin        derivatives for example etoposide or teniposide;    -   anti-tumour vinca alkaloids for example vinblastine, vincristine        or vinorelbine;    -   anti-tumour nucleoside derivatives for example 5-fluorouracil,        gemcitabine or capecitabine;    -   alkylating agents such as nitrogen mustard or nitrosourea for        example cyclophosphamide, chlorambucil, carmustine or lomustine;    -   anti-tumour anthracycline derivatives for example daunorubicin,        doxorubicin, idarubicin or mitoxantrone;    -   HER2 antibodies for example trastuzumab;    -   estrogen receptor antagonists or selective estrogen receptor        modulators for example tamoxifen, toremifene, droloxifene,        faslodex or raloxifene;    -   aromatase inhibitors such as exemestane, anastrozole, letrazole        and vorozole;    -   differentiating agents such as retinoids, vitamin D and retinoic        acid metabolism blocking agents (RAMBA) for example accutane;    -   DNA methyl transferase inhibitors for example azacytidine;    -   kinase inhibitors for example flavoperidol, imatinib mesylate or        gefitinib;    -   farnesyltransferase inhibitors;    -   other HDAC inhibitors;    -   inhibitors of the ubiquitin-proteasome pathway for example        Velcade; or    -   Yondelis.

The term “platinum coordination compound” is used herein to denote anytumour cell growth inhibiting platinum coordination compound whichprovides platinum in the form of an ion.

The term “taxane compounds” indicates a class of compounds having thetaxane ring system and related to or derived from extracts from certainspecies of yew (Taxus) trees.

The term “topisomerase inhibitors” is used to indicate enzymes that arecapable of altering DNA topology in eukaryotic cells. They are criticalfor important cellular functions and cell proliferation. There are twoclasses of topoisomerases in eukaryotic cells, namely type I and typeII. Topoisomerase I is a monomeric enzyme of approximately 100,000molecular weight. The enzyme binds to DNA and introduces a transientsingle-strand break, unwinds the double helix (or allows it to unwind)and subsequently reseals the break before dissociating from the DNAstrand. Topisomerase II has a similar mechanism of action which involvesthe induction of DNA strand breaks or the formation of free radicals.

The term “camptothecin compounds” is used to indicate compounds that arerelated to or derived from the parent camptothecin compound which is awater-insoluble alkaloid derived from the Chinese tree Camptothecinacuminata and the Indian tree Nothapodytes foetida.

The term “podophyllotoxin compounds” is used to indicate compounds thatare related to or derived from the parent podophyllotoxin, which isextracted from the mandrake plant.

The term “anti-tumour vinca alkaloids” is used to indicate compoundsthat are related to or derived from extracts of the periwinkle plant(Vinca rosea).

The term “alkylating agents” encompass a diverse group of chemicals thathave the common feature that they have the capacity to contribute, underphysiological conditions, alkyl groups to biologically vitalmacromolecules such as DNA. With most of the more important agents suchas the nitrogen mustards and the nitrosoureas, the active alkylatingmoieties are generated in vivo after complex degradative reactions, someof which are enzymatic. The most important pharmacological actions ofthe alkylating agents are those that disturb the fundamental mechanismsconcerned with cell proliferation in particular DNA synthesis and celldivision. The capacity of alkylating agents to interfere with DNAfunction and integrity in rapidly proliferating tissues provides thebasis for their therapeutic applications and for many of their toxicproperties.

The term “anti-tumour anthracycline derivatives” comprise antibioticsobtained from the fungus Strep. peuticus var. caesius and theirderivatives, characterised by having a tetracycline ring structure withan unusual sugar, daunosamine, attached by a glycosidic linkage.

Amplification of the human epidermal growth factor receptor 2 protein(HER 2) in primary breast carcinomas has been shown to correlate with apoor clinical prognosis for certain patients. Trastuzumab is a highlypurified recombinant DNA-derived humanized monoclonal IgG1 kappaantibody that binds with high affiniity and specificity to theextracellular domain of the HER2 receptor.

Many breast cancers have estrogen receptors and growth of these tumourscan be stimulated by estrogen. The terms “estrogen receptor antagonists”and “selective estrogen receptor modulators” are used to indicatecompetitive inhibitors of estradiol binding to the estrogen receptor(ER). Selective estrogen receptor modulators, when bound to the ER,induces a change in the three-dimensional shape of the receptor,modulating its binding to the estrogen responsive element (ERE) on DNA.

In postmenopausal women, the principal source of circulating estrogen isfrom conversion of adrenal and ovarian androgens (androstenedione andtestosterone) to estrogens (estrone and estradiol) by the aromataseenzyme in peripheral tissues. Estrogen deprivation through aromataseinhibition or inactivation is an effective and selective treatment forsome postmenopausal patients with hormone-dependent breast cancer.

The term “antiestrogen agent” is used herein to include not onlyestrogen receptor antagonists and selective estrogen receptor modulatorsbut also aromatase inhibitors as discussed above.

The term “differentiating agents” encompass compounds that can, invarious ways, inhibit cell proliferation and induce differentiation.Vitamin D and retinoids are known to play a major role in regulatinggrowth and differentiation of a wide variety of normal and malignantcell types. Retinoic acid metabolism blocking agents (RAMBA's) increasethe levels of endogenous retinoic acids by inhibiting the cytochromeP450-mediated catabolism of retinoic acids.

DNA methylation changes are among the most common abnormalities in humanneoplasia. Hypermethylation within the promotors of selected genes isusually associated with inactivation of the involved genes. The term“DNA methyl transferase inhibitors” is used to indicate compounds thatact through pharmacological inhibition of DNA methyl transferase andreactivation of tumour suppressor gene expression.

The term “kinase inhibitors” comprises potent inhibitors of kinases thatare involved in cell cycle progression and programmed cell death(apoptosis).

The term “farnesyltransferase inhibitors” is used to indicate compoundsthat were designed to prevent farnesylation of Ras and otherintracellular proteins. They have been shown to have effect on malignantcell proliferation and survival.

The term “other HDAC inhibitors” comprises but is not limited to:

-   -   carboxylates for example butyrate, cinnamic acid,        4-phenylbutyrate or valproic acid;    -   hydroxamic acids for example suberoylanilide hydroxamic acid        (SAHA), piperazine containing SAHA analogues, biaryl hydroxamate        A-161906 and its carbozolylether-, tetrahydropyridine- and        tetralone-analogues, bicyclic aryl-N-hydroxycarboxamides,        pyroxamide, CG-1521, PXD-101, sulfonamide hydroxamic acid,        LAQ-824, LBH-589, trichostatin A (TSA), oxamflatin, scriptaid,        scriptaid related tricyclic molecules, m-carboxy cinnamic acid        bishydroxamic acid (CBHA), CBHA-like hydroxamic acids,        trapoxin-hydroxamic acid analogue, R306465 and related benzoyl-        and heteroaryl-hydroxamic acids, aminosuberates and        malonyldiamides;    -   cyclic tetrapeptides for example trapoxin, apidicin,        depsipeptide, spiruchostatin-related compounds, RedFK-228,        sulfhydryl-containing cyclic tetrapeptides (SCOPs), hydroxamic        acid containing cyclic tetrapeptides (CHAPs), TAN-174s and        azumamides;    -   benzamides for example MS-275 or CI-994, or    -   depudecin.

The term “inhibitors of the ubiquitin-proteasome pathway” is used toidentify compounds that inhibit the targeted destruction of cellularproteins in the proteasome, including cell cycle regulatory proteins.

For the treatment of cancer the compounds according to the presentinvention may be administered to a patient as described above, inconjunction with irradiation. Irradiation means ionising radiation andin particular gamma radiation, especially that emitted by linearaccelerators or by radionuclides that are in common use today. Theirradiation of the tumour by radionuclides can be external or internal.

The present invention also relates to a combination according to theinvention of an anti-cancer agent and a HDAC inhibitor according to theinvention.

The present invention also relates to a combination according to theinvention for use in medical therapy for example for inhibiting thegrowth of tumour cells.

The present invention also relates to a combinations according to theinvention for inhibiting the growth of tumour cells.

The present invention also relates to a method of inhibiting the growthof tumour cells in a human subject which comprises administering to thesubject an effective amount of a combination according to the invention.

This invention further provides a method for inhibiting the abnormalgrowth of cells, including transformed cells, by administering aneffective amount of a combination according to the invention.

The other medicinal agent and HDAC inhibitor may be administeredsimultaneously (e.g. in separate or unitary compositions) orsequentially in either order. In the latter case, the two compounds willbe administered within a period and in an amount and manner that issufficient to ensure that an advantageous or synergistic effect isachieved. It will be appreciated that the preferred method and order ofadministration and the respective dosage amounts and regimes for eachcomponent of the combination will depend on the particular othermedicinal agent and HDAC inhibitor being administered, their route ofadministration, the particular tumour being treated and the particularhost being treated. The optimum method and order of administration andthe dosage amounts and regime can be readily determined by those skilledin the art using conventional methods and in view of the information setout herein.

The platinum coordination compound is advantageously administered in adosage of 1 to 500 mg per square meter (mg/m²) of body surface area, forexample 50 to 400 mg/m², particularly for cisplatin in a dosage of about75 mg/m² and for carboplatin in about 300 mg/m² per course of treatment.

The taxane compound is advantageously administered in a dosage of 50 to400 mg per square meter (mg/m²) of body surface area, for example 75 to250 mg/m², particularly for paclitaxel in a dosage of about 175 to 250mg/m² and for docetaxel in about 75 to 150 mg/m² per course oftreatment.

The camptothecin compound is advantageously administered in a dosage of0.1 to 400 mg per square meter (mg/m²) of body surface area, for example1 to 300 mg/m², particularly for irinotecan in a dosage of about 100 to350 mg/m² and for topotecan in about 1 to 2 mg/m² per course oftreatment.

The anti-tumour podophyllotoxin derivative is advantageouslyadministered in a dosage of 30 to 300 mg per square meter (mg/m²) ofbody surface area, for example 50 to 250 mg/m², particularly foretoposide in a dosage of about 35 to 100 mg/m² and for teniposide inabout 50 to 250 mg/m² per course of treatment.

The anti-tumour vinca alkaloid is advantageously administered in adosage of 2 to 30 mg per square meter (mg/m²) of body surface area,particularly for vinblastine in a dosage of about 3 to 12 mg/m², forvincristine in a dosage of about 1 to 2 mg/m², and for vinorelbine indosage of about 10 to 30 mg/m² per course of treatment.

The anti-tumour nucleoside derivative is advantageously administered ina dosage of 200 to 2500 mg per square meter (mg/m²) of body surfacearea, for example 700 to 1500 mg/m², particularly for 5-FU in a dosageof 200 to 500 mg/m², for gemcitabine in a dosage of about 800 to 1200mg/m² and for capecitabine in about 1000 to 2500 mg/m² per course oftreatment.

The alkylating agents such as nitrogen mustard or nitrosourea isadvantageously administered in a dosage of 100 to 500 mg per squaremeter (mg/m²) of body surface area, for example 120 to 200 mg/m²,particularly for cyclophosphamide in a dosage of about 100 to 500 mg/m²,for chlorambucil in a dosage of about 0.1 to 0.2 mg/kg, for carmustinein a dosage of about 150 to 200 mg/m², and for lomustine in a dosage ofabout 100 to 150 mg/m² per course of treatment.

The anti-tumour anthracycline derivative is advantageously administeredin a dosage of 10 to 75 mg per square meter (mg/m²) of body surfacearea, for example 15 to 60 mg/m², particularly for doxorubicin in adosage of about 40 to 75 mg/m², for daunorubicin in a dosage of about 25to 45 mg/m², and for idarubicin in a dosage of about 10 to 15 mg/m² percourse of treatment.

Trastuzumab is advantageously administered in a dosage of 1 to 5 mg persquare meter (mg/m²) of body surface area, particularly 2 to 4 mg/m² percourse of treatment.

The antiestrogen agent is advantageously administered in a dosage ofabout 1 to 100 mg daily depending on the particular agent and thecondition being treated. Tamoxifen is advantageously administered orallyin a dosage of 5 to 50 mg, preferably 10 to 20 mg twice a day,continuing the therapy for sufficient time to achieve and maintain atherapeutic effect. Toremifene is advantageously administered orally ina dosage of about 60 mg once a day, continuing the therapy forsufficient time to achieve and maintain a therapeutic effect.Anastrozole is advantageously administered orally in a dosage of about 1mg once a day. Droloxifene is advantageously administered orally in adosage of about 20-100 mg once a day. Raloxifene is advantageouslyadministered orally in a dosage of about 60 mg once a day. Exemestane isadvantageously administered orally in a dosage of about 25 mg once aday.

These dosages may be administered for example once, twice or more percourse of treatment, which may be repeated for example every 7, 14, 21or 28 days.

In view of their useful pharmacological properties, the components ofthe combinations according to the invention, i.e. the other medicinalagent and the HDAC inhibitor may be formulated into variouspharmaceutical forms for administration purposes. The components may beformulated separately in individual pharmaceutical compositions or in aunitary pharmaceutical composition containing both components.

The present invention therefore also relates to a pharmaceuticalcomposition comprising the other medicinal agent and the HDAC inhibitortogether with one or more pharmaceutical carriers.

The present invention also relates to a combination according to theinvention in the form of a pharmaceutical composition comprising ananti-cancer agent and a HDAC inhibitor according to the inventiontogether with one or more pharmaceutical carriers.

The present invention further relates to the use of a combinationaccording to the invention in the manufacture of a pharmaceuticalcomposition for inhibiting the growth of tumour cells.

The present invention further relates to a product containing as firstactive ingredient a HDAC inhibitor according to the invention and assecond active ingredient an anticancer agent, as a combined preparationfor simultaneous, separate or sequential use in the treatment ofpatients suffering from cancer.

EXPERIMENTAL PART

The following examples are provided for purposes of illustration.

Hereinafter, “DCM” is defined as dichloromethane, “DIPE” is defined asdiisopropyl ether, “DMA” is defined as is defined asN,N-dimethylacetamide, “DMSO” is defined as dimethylsulfoxide, “EDC” isdefined as N′-(ethylcarbonimidoyl)-N,N-dimethyl-1,3-propanediamine,monohydrochloride, “EtOAc” is defined as ethyl acetate, “EtOH” isdefined as ethanol, “HOBt” is defined as 1-hydroxy-1H-benzotriazole,“MeOH” is defined as methanol, “TFA” is defined as trifluoroacetic acidand “THF” is defined as tetrahydrofuran.

A. Preparation of the Intermediate Compounds Example A1 a) Preparationof Intermediate 1

A mixture of 2-[4-(aminomethyl)-1-piperidinyl]-5-pyrimidinecarboxylicacid, ethyl ester (0.0114 mol), 1-methyl-1H-indole-3-carboxaldehyde(0.017 mol) and MgSO₄ (0.5 g) in MeOH (80 ml) was stirred and refluxedfor 15 hours, then cooled to room temperature. Sodium tetrahydroborate(0.018 mol) was added portionwise. The mixture was stirred at roomtemperature 5 hours, poured out into water and extracted with EtOAc. Theorganic layer was separated, dried (MgSO₄), filtered, and the solventwas evaporated. The residue (6.6 g) was purified by columnchromatography over silica gel (15-40 μm) (eluent: DCM/MeOH/NH₄OH94/6/0.5). The pure fractions were collected and the solvent wasevaporated, yielding 4.3 g (90%) of intermediate 1.

b) Preparation of Intermediate 2

A mixture of intermediate 1 (0.0037 mol) and sodium hydroxide (0.0074mol) in EtOH (60 ml) was stirred at 50° C. for 15 hours, then cooled toroom temperature and the solvent was evaporated till dryness, yielding1.5 g (100%) of intermediate 2.

c) Preparation of Intermediate 3

EDC (0.0075 mol), then HOBt (0.0075 mol) then0-(tetrahydro-2H-pyran-2-yl)-hydroxylamine (0.015 mol) were added atroom temperature to a mixture of intermediate 2 (0.005 mol) in DCM (100ml) and THF (100 ml) under N₂ flow. The mixture was stirred at 40° C.for 4 hours, poured out into ice water and extracted with DCM. Theorganic layer was separated, dried (MgSO₄), filtered, and the solventwas evaporated. The residue (4 g) was purified by column chromatographyover silica gel (15-40 μm) (eluent: DCM/MeOH/NH₄OH 96/4/0.5). The purefractions were collected and the solvent was evaporated, yielding 1 g(42%) of intermediate 3. A fraction (0.051 g) was crystallized fromDIPE. The precipitate was filtered off and dried, yielding 0.03 g ofintermediate 3, melting point 70° C.

Example A2 a) Preparation of Intermediate 4

A mixture of 2-[4-(aminomethyl)-1-piperidinyl]-5-pyrimidinecarboxylicacid, ethyl ester (0.0072 mol) in THF (40 ml) and sodium hydroxide 1N(40 ml) was stirred at room temperature overnight. Hydrochloric acid 1N(40 ml) was added. The mixture was stirred for 10 minutes. Sodiumcarbonate (0.0216 mol) was added. The mixture was stirred for 10minutes. 1-[[(9H-fluoren-9-ylmethoxy)carbonyl]oxy]-2,5-pyrrolidinedione(0.0072 mol) was added portionwise. The mixture was stirred at roomtemperature for 6 hours, then cooled to 0° C. and acidified withhydrochloric acid. The precipitate was filtered, washed with diethylether and dried, yielding 4.1 g (100%) of intermediate 4.

b) Preparation of Intermediate 5

Triethylamine (0.02 mol), EDC (0.0082 mol) and HOBt (0.0082 mol) wereadded at room temperature to a mixture of intermediate 4 (0.0068 mol) inDCM/THF (200 ml) under N₂ flow. The mixture was stirred at roomtemperature for 15 minutes. O-(tetrahydro-2H-pyran-2-yl)-hydroxylamine(0.0082 mol) was added. The mixture was stirred at room temperature for48 hours, poured out into water and extracted with DCM. The organiclayer was washed with NaHCO₃ 10%, dried (MgSO₄), filtered, and thesolvent was evaporated. The residue (4 g) was purified by columnchromatography over silica gel (15-40 μm) (eluent: DCM/MeOH/NH₄OH98/2/0.1). The pure fractions were collected and the solvent wasevaporated, yielding 3.4 g (89%) of intermediate 5.

c) Preparation of Intermediate

A mixture of intermediate 5 (0.0355 mol) and piperidine (0.089 mol) inDCM (400 ml) was stirred at 35° C. during 72 hours. The solvent wasevaporated. The residue was purified by column chromatography oversilica gel (15-40 μm) (eluent: DCM/MeOH/NH₄OH 80/20/2). The purefractions were collected and the solvent was evaporated, yielding 6.7 g(56%). Part of the residue (0.79 g) was crystallized from diethyl ether.The precipitate was filtered off and dried, yielding 0.62 g ofintermediate 6, melting point 129° C.

d) Preparation of Intermediate 7

A mixture of intermediate 6 (0.0009 mol) and5-chloro-1H-indole-3-carboxaldehyde (0.0012 mol) in 1,2-dichloro-ethane(30 ml) was stirred at room temperature overnight.Tris(acetato-α-O)hydro-borate(1-), sodium (0.0013 mol) was addedportionwise. The mixture was stirred at room temperature for 4 hours,poured out into water/NaOH 3N and extracted with DCM. The organic layerwas separated, dried (MgSO₄), filtered, and the solvent was evaporated.The residue (0.5 g) was purified by column chromatography over silicagel (15-40 μm) (eluent DCM/MeOH/NH₄OH 93/7/0.5). Two fractions werecollected and the solvent was evaporated, yielding 0.07 g (16%) ofintermediate 7.

Example A3 a) Preparation of Intermediate 8

A solution of 2-(methylsulfonyl)-5-pyrimidinecarboxylic acid, ethylester (0.094 mol) in acetonitrile (40 ml) was added at 10° C. to asuspension of 4-piperidinemethanol (0.086 mol) and potassium carbonate(0.172 mol) in acetonitrile (200 ml) under N₂ flow. The mixture wasbrought to room temperature, then stirred for 4 hours, poured out intowater and extracted with EtOAc. The organic layer was separated, dried(MgSO₄), filtered, and the solvent was evaporated. The residue (23 g)was crystallized from acetonitrile/diethyl ether. The precipitate wasfiltered off and dried, yielding 7.8 g (34%) of intermediate 8. Themother layer was evaporated. The residue (17 g) was purified by columnchromatography over silica gel (20-45 μm) (eluent: DCM/MeOH/NH₄OH97/3/0.1). The pure fractions were collected and the solvent wasevaporated, yielding 4.6 g (20%) of intermediate 8, melting point 129°C.

b) Preparation of Intermediate 9

Triethylamine (0.038 mol) then methanesulfonyl chloride (0.025 mol) wereadded at 0° C. to a solution of intermediate 8 (0.0189 mol) in DCM (80ml) under N₂ flow. The mixture was stirred at 0° C. for 2 hours andpoured out into ice water. The organic layer was separated, dried(MgSO₄), filtered, and the solvent was evaporated, yielding 6.5 g (100%)of intermediate 9.

c) Preparation of Intermediate 10

A mixture of intermediate 9 (0.0189 mol),N-methyl-1H-indole-3-ethanamine (0.0172 mol) and potassium carbonate(0.0344 mol) in acetonitrile (180 ml) was stirred and refluxed for 24hours, poured out into water and extracted with EtOAc. The organic layerwas separated, dried (MgSO₄), filtered, and the solvent was evaporated.The residue (8.5 g) was purified by column chromatography over silicagel (70-200 μm) (eluent: DCM/MeOH/NH₄OH 98/2/0 to 97/3/0.1). The purefractions were collected and the solvent was evaporated, yielding 1.25 g(20%) of intermediate 10.

d) Preparation of Intermediate 11

A mixture of intermediate 10 (0.003 mol) and sodium hydroxide (0.006mol) in EtOH (80 ml) was stirred and refluxed overnight, then cooled toroom temperature and the solvent was evaporated till dryness, yielding1.3 g (100%) of intermediate 11, mp. >260° C.

e) Preparation of Intermediate 12

EDC (0.0045 mol) then HOBt (0.0045 mol) were added at room temperatureto a mixture of intermediate 11 (0.003 mol) in THF (100 ml) and DCM (100ml) under N₂ flow. The mixture was stirred at room temperature for 15minutes. O-(tetrahydro-2H-pyran-2-yl)-hydroxylamine (0.012 mol) wasadded. The mixture was stirred at room temperature for 72 hours, pouredout into water and extracted with DCM. The organic layer was separated,dried (MgSO₄), filtered, and the solvent was evaporated. The residue (3g) was purified by column chromatography over silica gel (15-40μm)(eluent: DCM/MeOH/NH₄OH 94/6/0.1;). The pure fractions were collectedand the solvent was evaporated. The residue (0.82 g) was taken up indiethyl ether. The precipitate was filtered off and dried, yielding 0.78g of intermediate 12, melting point 154° C.

Example A4 a) Preparation of Intermediate 13

A mixture of 2-[4-(aminomethyl)-1-piperidinyl]-5-pyrimidinecarboxylicacid, ethyl ester (0.0049 mol), 1H-indole-3-ethanol, methanesulfonate(ester) (0.0054 mol) and potassium carbonate (0.01 mol) in acetonitrile(20 ml) was stirred and refluxed overnight, then cooled, poured out intoice water and extracted with DCM. The organic layer was separated, dried(MgSO₄), filtered, and the solvent was evaporated. The residue (2.2 g)was purified by column chromatography over silica gel (15-40 μm)(eluent: DCM/MeOH/NH₄OH 96/4/0.2). The pure fractions were collected andthe solvent was evaporated, yielding 0.442 g (22%) of intermediate 13,melting point 238° C.

b) Preparation of Intermediate 14

A mixture of intermediate 13 (0.0025 mol),(2-bromoethoxy)(1,1-dimethylethyl)dimethyl-silane (0.0034 mol) andN-ethyl-N-(1-methylethyl)-2-propanamine (0.0038 mol) in DMSO (20 ml) wasstirred at 50° C. for 15 hours, then cooled to room temperature, pouredout into ice water and extracted with DCM. The organic layer wasseparated, dried (MgSO₄), filtered, and the solvent was evaporated. Theresidue (1.7 g) was purified by column chromatography over silica gel(15-40 μm) (eluent: DCM/MeOH/NH₄OH 98/2/0.1). The pure fractions werecollected and the solvent was evaporated, yielding 0.76 g (54%) ofintermediate 14.

c) Preparation of Intermediate 15

A mixture of intermediate 14 (0.0013 mol) and sodium hydroxide (0.0027mol) in EtOH (40 ml) was stirred at 80° C. overnight, then cooled toroom temperature and the solvent was evaporated, yielding 0.75 g (100%)of intermediate 15.

d) Preparation of Intermediate

EDC (0.002 mol) then HOBt (0.002 mol) were added at room temperature toa mixture of intermediate 15 (0.0013 mol) in THF (80 ml) and DCM (80 ml)under N₂ flow. The mixture was stirred at room temperature for 15minutes. O-(tetrahydro-2H-pyran-2-yl)-hydroxylamine (0.0068 mol) wasadded. The mixture was stirred at room temperature for 72 hours, pouredout into water and extracted with DCM. The organic layer was separated,dried (MgSO₄), filtered, and the solvent was evaporated. The residue(1.3 g) was purified by column chromatography over silica gel (15-40μm)(eluent: DCM/MeOH/NH₄OH 95/5/0.1). The pure fractions were collectedand the solvent was evaporated, yielding 0.38 g of intermediate 16.

e) Preparation of Intermediate 17

A mixture of intermediate 16 (0.0011 mol) and tetrabutylammoniumfluoride (0.0032 mol) in THF (10 ml) was stirred at room temperature for72 hours, poured out into water and extracted with EtOAc. The organiclayer was washed with water, dried (MgSO₄), filtered and the solvent wasevaporated, yielding 0.5 g (88%) of intermediate 17.

Example A5 a) Preparation of Intermediate 45

A solution of 2-chloro-5-pyrimidinecarboxylic acid, methyl ester (0.058mol) in DMA (80 ml) was added dropwise to a solution of4-piperidinemethanamine (0.116 mol) and N-ethyldiisopropylamine (0.145mol) in DMA (150 ml) under N₂ flow. The mixture was stirred at roomtemperature for 1 hour and 30 minutes, poured out into ice water andextracted with EtOAc, then with DCM. The organic layer was washed withwater, dried (MgSO₄), filtered and the solvent was evaporated. Theresidue was crystallized from DIPE. The precipitate was filtered off anddried, yielding 10 g (65%) of intermediate 45.

b) Preparation of Intermediate 46

A mixture of intermediate 45 (0.0024 mol),1-methyl-5-nitro-1H-indole-3-carboxaldehyde (0.0036 mol) and MgSO₄ (0.25g) in MeOH (80 ml) was stirred at 60° C. overnight, then cooled to roomtemperature. Sodium tetrahydroborate (0.0041 mol) was added portionwise.The mixture was stirred at room temperature for 18 hours, poured outinto water and extracted with EtOAc. The organic layer was separated,dried (MgSO₄), filtered and the solvent was evaporated. The residue (1.1g) was crystallized from acetonitrile. The precipitate was filtered offand dried, yielding 0.9 g (86%) of intermediate 46, melting point:150°C.

c) Preparation of Intermediate 47

A mixture of intermediate 46 (0.002 mol) and sodium hydroxide (0.008mol) in EtOH (60 ml) was stirred at 60° C. overnight, then cooled toroom temperature and evaporated. The residue was taken up in diethylether. The precipitate was filtered off and dried, yielding 0.6 g (67%)of intermediate 47.

d) Preparation of Intermediate 48

EDC (0.0019 mol) and HOBt (0.0019 mol) were added at room temperature toa solution of intermediate 47 (0.0013 mol) and triethylamine (0.0039mol) in DCM/THF (50/50) (100 ml) under N₂ flow. The mixture was stirredat room temperature for 15 minutes.O-(tetrahydro-2H-pyran-2-yl)-hydroxylamine (0.0026 mol) was added. Themixture was stirred at room temperature for 72 hours, poured out intowater and extracted with DCM. The organic layer was separated, dried(MgSO₄), filtered and the solvent was evaporated. The residue (1 g) waspurified by column chromatography over kromasil (5 μm) (eluent:DCM/MeOH/NH₄OH 98/2/0.2 to 92/8/0.2). The pure fractions were collectedand the solvent was evaporated, yielding 0.101 g (15%) of intermediate48.

Example A6 a) Preparation of Intermediate 49

A solution of 2-chloro-5-pyrimidinecarboxylic acid, methyl ester (0.033mol) in DCM (80 ml) was added at room temperature to a solution of4-piperidinemethanol (0.066 mol) and N-ethyldiisopropylamine (0.083 mol)in DCM (100 ml) under N₂ flow. The mixture was stirred at roomtemperature for 3 hours and 30 minutes, poured out into ice water andextracted with DCM. The organic layer was separated, dried (MgSO₄),filtered and the solvent was evaporated. The residue was taken up inpentane. The precipitate was filtered off and dried, yielding 7.88 g(95%) of intermediate 49.

b) Preparation of Intermediate 50

DMSO (0.058 mol) was added dropwise at −78° C. to a solution ofethanedioyl dichloride (0.0278 mol) in DCM (50 ml) under N₂ flow. Themixture was stirred for 15 minutes. A solution of intermediate 49 (0.023mol) in DCM (200 ml) was added dropwise. The mixture was stirred at −78°C. for 1 hour and 30 minutes. Triethylamine (0.118 mol) was addeddropwise. The mixture was stirred at −78° C. for 1 hour, poured out intowater and extracted with DCM. The organic layer was separated, dried(MgSO₄), filtered and the solvent was evaporated. The residue wascrystallized from diethyl ether. The precipitate was filtered off anddried, yielding 3.06 g (54%) of intermediate 50.

c) Preparation of Intermediate 51

Intermediate 50 (0.0122 mol) was added at 5° C. to a solution of1-methyl-1H-indole-3-ethanamine (0.0122 mol) in MeOH (270 ml) under N₂flow. The mixture was stirred a few minutes. Sodiumcyanoborohydride(0.0183 mol) and acetic acid (0.0183 mol) were added. The mixture wasstirred at room temperature for 48 hours, poured out into potassiumcarbonate 10% and extracted with DCM. The organic layer was separated,dried (MgSO₄), filtered and the solvent was evaporated. The residue (4.9g) was purified by column chromatography over silica gel (15-40 μm)(eluent: DCM/MeOH/NH₄OH 97/3/0.1). The pure fractions were collected andthe solvent was evaporated, yielding 1.2 g (24%) of intermediate 51.

d) Preparation of Intermediate 52

A mixture of intermediate 51 (0.0009 mol) and sodium hydroxide (0.0039mol) in EtOH (60 ml) was stirred and refluxed for 15 hours, thenevaporated till dryness, yielding intermediate 52. This intermediate wasused directly in the next reaction step.

e) Preparation of Intermediate

HOBt (0.0019 mol) then EDC (0.0019 mol) were added at room temperatureto a solution of intermediate 52 (0.0009 mol) andO-(tetrahydro-2H-pyran-2-yl)-hydroxylamine (0.0019 mol) in DCM/THF (130ml). The mixture was stirred at room temperature for 48 hours, pouredout into water and extracted with DCM. The organic layer was separated,dried (MgSO₄), filtered and the solvent was evaporated. The residue(0.93 g) was purified by column chromatography over silica gel (15-40μm) (eluent: DCM/MeOH/NH₄OH 97/3/0.1). The pure fractions were collectedand the solvent was evaporated, yielding 0.155 g (33%) of intermediate53.

Example A7 a) Preparation of Intermediate 54

A mixture of 2-[4-(aminomethyl)-1-piperidinyl]-5-pyrimidinecarboxylicacid, ethyl ester (0.0038 mol), 1-ethyl-1H-indole-3-carboxaldehyde(0.0049 mol) and Pd/C 10% (0.5 g) in MeOH (20 ml) containing 1 ml of a10% thiopene solution in EtOH, was hydrogenated at room temperature for24 hours under a 3 bar pressure, then filtered over celite. The solventwas evaporated till dryness. The residue (1.8 g) was purified by columnchromatography over silica gel (15-40 nm) (eluent: DCM/MeOH/NH₄OH95/5/0.2 to 93/7/0.5). The pure fractions were collected and the solventwas evaporated, yielding 0.7 g (44%) of intermediate 54.

b) Preparation of Intermediate 55

Sodium hydride 60% (0.009 mol) was added at 0° C. to a solution ofintermediate 54 (0.0045 mol) in THF (50 ml) under N₂ flow. The mixturewas stirred at room temperature for 1 hour. A solution of iodo-ethane(0.0062 mol) in THF (10 ml) was added dropwise. The mixture was stirredat room temperature overnight, poured out into water and extracted withEtOAc. The organic layer was separated, dried (MgSO₄), filtered, and thesolvent was evaporated. The residue (0.6 g) was purified by columnchromatography over silica gel (15-40 μm) (eluent: DCM/MeOH/NH₄OH95/5/0.1). The pure fractions were collected and the solvent wasevaporated, yielding 0.16 g (8%) of intermediate 55.

c) Preparation of Intermediate 56

A mixture of intermediate 55 (0.0003 mol) and sodium hydroxide (0.03 g)in EtOH (15 ml) was stirred at 80° C. for 6 hours, then evaporated tilldryness, yielding 0.16 g (100%) of intermediate 56.

d) Preparation of Intermediate 57

EDC (0.0005 mol) and HOBt (0.0005 mol) were added at room temperature toa mixture of intermediate 56 (0.0003 mol) in DCM (20 ml) and THF (20 ml)under N₂ flow. The mixture was stirred for 15 minutes.O-(tetrahydro-2H-pyran-2-yl)-hydroxylamine (0.0007 mol) was added. Themixture was stirred at room temperature for 72 hours, poured out intowater and extracted with DCM. The organic layer was separated, dried(MgSO₄), filtered, and the solvent was evaporated. The residue (0.3 g)was purified by column chromatography over kromasil (5 μm) (eluent:DCM/MeOH/NH₄OH 93/7/0.35). The pure fractions were collected and thesolvent was evaporated, yielding 0.03 g (16%) of intermediate 57.

Example A8 a) Preparation of Intermediate 58

Sodium hydride (0.011 mol) was added at 5° C. to a solution ofintermediate 13 (0.0037 mol) in THF (30 ml) under N₂ flow. The mixturewas stirred for 30 minutes. A solution of iodomethane (0.0081 mol) inTHF (10 ml) was added dropwise. The mixture was stirred at 10° C. for 2hours, then brought to room temperature for 1 hour and 30 minutes,poured out into ice water and extracted with DCM. The organic layer wasseparated, dried (MgSO₄), filtered, and the solvent was evaporated. Theresidue (1.7 g) was purified by column chromatography over kromasil(15-40 μm) (eluent: DCM/MeOH/NH₃OH 98/2/0.1). Two fractions werecollected and the solvent was evaporated, yielding 0.265 g ofintermediate 58 and 0.57 g (17%) of intermediate 10.

b) Preparation of Intermediate 59

A mixture of intermediate 58 (0.0006 mol) and sodium hydroxide (0.0012mol) in EtOH (30 ml) was stirred at 80° C. overnight, then cooled toroom temperature and the solvent was evaporated, yielding 0.26 g (100%)of intermediate 59.

c) Preparation of Intermediate 60

EDC (0.0009 mol) and HOBt (0.0009 mol) were added at room temperature toa solution of intermediate 59 (0.0006 mol) in THF (30 ml) and DCM (30ml) under N₂ flow. The mixture was stirred at room temperature for 15minutes. O-(tetrahydro-2H-pyran-2-yl)-hydroxylamine (0.0012 mol) wasadded. The mixture was stirred at room temperature for 24 hours, pouredout into water and extracted with DCM. The organic layer was separated,dried (MgSO₄), filtered, and the solvent was evaporated. The residue(0.6 g) was purified by column chromatography over kromasil (5 μm)(eluent: DCM/MeOH/NH₄OH 99/1/0.05 and 94/6/0.3). The pure fractions werecollected and the solvent was evaporated, yielding 0.1 g (33%) ofintermediate 60.

Example A9 a) Preparation of Intermediate 61

DMSO (0.127 mol) was added at −78° C. to a solution of ethanedioyldichloride (0.061 mol) in DCM (110 ml) under N₂ flow. The mixture wasstirred for 15 minutes. A solution of intermediate 8 (0.051 mol) in DCM(200 ml) was added. The mixture was stirred at −78° C. for 1 hour and 30minutes. Triethylamine (0.26 mol) was added dropwise. The mixture wasstirred at −78° C. for 15 minutes, then brought to room temperature for2 hours and 30 minutes. Water was added. The mixture was extracted withDCM. The organic layer was separated, dried (MgSO₄), filtered, and thesolvent was evaporated. The residue (14 g) was purified by columnchromatography over silica gel (20-45 μm) (eluent: cyclohexane/EtOAc70/30). The pure fractions were collected and the solvent wasevaporated, yielding 7.6 g (57%) of intermediate 61.

b) Preparation of Intermediate 62

Sodiumcyanoborohydride (0.049 mol) and acetic acid (0.034 ml) were addedat room temperature to a solution of5-chloro-1-methyl-1H-Indole-3-ethanamine (0.031 mol) and intermediate 61(0.034 mol) in MeOH (700 ml) under N₂ flow. The mixture was stirred atroom temperature for 24 hours, poured out into potassium carbonate 10%and extracted with DCM. The organic layer was separated, dried (MgSO₄),filtered and the solvent was evaporated. The residue (14.8 g) waspurified by column chromatography over silica gel (20-45 μm) (eluent:DCM/MeOH/NH₄OH 95/5/0.2). The pure fractions were collected and thesolvent was evaporated, yielding 4.52 g (32%) of intermediate 62.

c) Preparation of Intermediate 63

Methanesulfonyl chloride (0.0049 mol) was added at 5° C. to a solutionof intermediate 62 (0.004 mol) and triethylamine (0.008 mol) in DCM (150ml) under N₂ flow. The mixture was stirred at room temperature for 24hours, poured out into ice water and extracted with DCM. The organiclayer was separated, dried (MgSO₄), filtered and the solvent wasevaporated. The residue (2.39 g) was taken up in DIPE. The precipitatewas filtered off and dried, yielding 1.78 g (84%) of intermediate 63,melting point 162° C.

d) Preparation of Intermediate

A mixture of intermediate 63 (0.0032 mol) and sodium hydroxide (0.0128mol) in EtOH (150 ml) was stirred and refluxed for 5 hours, then cooledto room temperature and taken up in diethyl ether. The precipitate wasfiltered off and dried, yielding 1.57 g (99%) of intermediate 64,melting point >260° C.

e) Preparation of Intermediate 65

EDC (0.0064 mol) and HOBt (0.0064 mol) were added at room temperature toa solution of intermediate 64 (0.0032 mol) in THF (160 ml) and DCM (160ml) under N₂ flow. The mixture was stirred at room temperature for 30minutes. O-(tetrahydro-2H-pyran-2-yl)-hydroxylamine (0.0064 mol) wasadded. The mixture was stirred at room temperature for 3 days, pouredout into water and extracted with DCM. The organic layer was separated,dried (MgSO₄), filtered and the solvent was evaporated. The residue(2.77 g) was purified by column chromatography over silica gel (15-40μm) (eluent: DCM/MeOH/NH₄OH 97/3/0.1). The pure fractions were collectedand the solvent was evaporated. The residue (0.385 g) was crystallizedfrom CH₃CN/diethyl ether. The precipitate was filtered off and dried,yielding 0.084 g of intermediate 65, melting point 179° C.

Example A10 a) Preparation of Intermediate 66

A solution of 2-(methylsulfonyl)-5-pyrimidinecarboxylic acid, ethylester (0.094 mol) in acetonitrile (240 ml) was added at room temperatureto a solution of 4-piperidinyl-carbamic acid, 1,1-dimethylethyl ester(0.078 mol) and potassium carbonate (0.156 mol) in acetonitrile (120 ml)under N₂ flow. The mixture was stirred at room temperature overnight,poured out into ice water and extracted with EtOAc. The organic layerwas washed with water, dried (MgSO₄), filtered, and the solvent wasevaporated. The residue was crystallized from diethyl ether. Theprecipitate was filtered off and dried, yielding 14.4 g (53%) ofintermediate 66, melting point 160° C.

b) Preparation of Intermediate

TFA (20 ml) was added at room temperature to a solution of intermediate66 (0.0225 mol) in DCM (110 ml). The mixture was stirred at roomtemperature for 15 hours, poured out into water and basified withpotassium carbonate. The mixture was extracted with DCM. The organiclayer was separated, dried (MgSO4), filtered, and the solvent wasevaporated, yielding 5.5 g (98%) of intermediate 67.

Example A11 a) Preparation of Intermediate 68

Sodium hydride 60% in oil (0.0069 mol) was added at 0° C. to a solutionof 2-methyl-1H-indole-3-acetic acid, ethyl ester (0.0046 mol) in THF (10ml) under N₂ flow. The mixture was stirred at room temperature for 1hour. Iodo-ethane (0.006 mol) was added. The mixture was stirred at roomtemperature for 18 hours and poured out into EtOAc and saturated NaCl.The organic layer was separated, dried (MgSO₄), filtered and the solventwas evaporated till dryness. The residue (1.1 g) was purified by columnchromatography over silica gel (15-40 μm) (eluent: cyclohexane/EtOAc80/20). The pure fractions were collected and the solvent wasevaporated, yielding 0.73 g (65%) of intermediate 68.

b) Preparation of Intermediate 69

A solution of diisobuytlaluminium hydride in toluene (0.0045 mol) wasadded dropwise at −78° C. to a solution of intermediate 68 (0.003 mol)in DCM (15 ml) and 1,2-dimethoxyethane (15 ml) (molecular sieves: 3angstrom) under N₂ flow. The mixture was stirred at −78° C. for 3 hours,then quenched with HCl 3N and extracted with DCM. The organic layer waswashed with water, dried (MgSO₄), filtered and the solvent wasevaporated till dryness, yielding 0.7 g (>100%) of intermediate 69.

c) Preparation of Intermediate 70

Titanium (IV) ethoxide (0.0023 mol) was added to a mixture ofintermediate 67 (0.0021 mol) and intermediate 69 (0.0021 mol) in1,2-dichloro-ethane (25 ml). The mixture was stirred at room temperaturefor 30 minutes. Tris(acetato-α-O)hydro-borate(1−), sodium (0.0023 mol)was added portionwise. The mixture was stirred at room temperature for18 hours, then quenched with NaHCO₃ and extracted with DCM. The organiclayer was separated, dried (MgSO₄), filtered and the solvent wasevaporated till dryness. The residue (1.2 g) was purified by columnchromatography over silica gel (Sum) (eluent: DCM/MeOH/NH₄OH 95/5/0.1).The pure fractions were collected and the solvent was evaporated,yielding 0.2 g (21%) of intermediate 70.

d) Preparation of Intermediate 71

A mixture of intermediate 70 (0.0004 mol) and sodium hydroxide (0.0009mol) in EtOH (30 ml) was stirred at 60° C. overnight, then cooled toroom temperature and evaporated, yielding 0.2 g (100%) of intermediate71.

e) Preparation of Intermediate 72

EDC (0.0007 mol) and HOBt (0.1 g) were added at room temperature to asolution of intermediate 71 (0.0004 mol) and triethylamine (0.0009 mol)in DCM/THF (40 ml) under N₂ flow. The mixture was stirred at roomtemperature for 15 minutes. O-(tetrahydro-2H-pyran-2-yl)-hydroxylamine(0.0009 mol) was added. The mixture was stirred at room temperature for72 hours, poured out into water and extracted with DCM. The organiclayer was separated, dried (MgSO₄), filtered and the solvent wasevaporated. The residue (0.4 g) was purified by column chromatographyover kromasil (5 μm)(eluent: DCM/MeOH/NH₄OH 98/2/0.1 to 90/10/1). Thepure fractions were collected and the solvent was evaporated, yielding0.087 g (37%) of intermediate 72.

Example A12 a) Preparation of Intermediate 73

Intermediate 50 (0.0046 mol) was added at 5° C. to a solution of6-methoxy-1-methyl-1H-Indole-3-ethanamine (0.0046 mol) in MeOH (100 ml)under N₂ flow. The mixture was stirred for 30 minutes.Sodiumcyanoborohydride (0.0068 mol) then acetic acid (0.0046 mol) wereadded portionwise. The mixture was stirred at room temperature for 48hours, poured out into potassium carbonate 10% and extracted with DCM.The organic layer was separated, dried (MgSO₄), filtered, and thesolvent was evaporated. The residue (4 g) was purified by columnchromatography over silica gel (15-40 μm)) (eluent: DCM/MeOH/NH₄OH96/4/0.2). The pure fractions were collected and the solvent wasevaporated. A part (0.7 g) of the residue (2.2 g) was crystallized fromacetonitrile. The precipitate was filtered off and dried, yielding 0.43g (61%) of intermediate 73, melting point 122° C.

b) Preparation of Intermediate 74

A mixture of intermediate 73 (0.0015 mol) and sodium hydroxide (0.006mol) in EtOH (90 ml) was stirred and refluxed for 8 hours, thenevaporated till dryness, yielding intermediate 74.

c) Preparation of Intermediate 75

HOBt (0.003 mol) then EDC (0.003 mol) were added at room temperature toa solution of intermediate 74 (0.0015 mol) andO-(tetrahydro-2H-pyran-2-yl)-hydroxylamine (0.003 mol) in THF/DCM (200ml) under N₂ flow. The mixture was stirred at room temperature for 48hours, poured out into water and extracted with DCM. The organic layerwas separated, dried (MgSO₄), filtered, and the solvent was evaporated.The residue (1.1 g) was purified by column chromatography over silicagel (10 μm) (eluent: DCM/MeOH/NH₄OH 95/5/0.5). The pure fractions werecollected and the solvent was evaporated. The residue (0.24 g) waspurified by column chromatography over silica gel (10 μm) (eluent:DCM/MeOH/NH₄OH 95/5/0.5). The pure fractions were collected and thesolvent was evaporated, yielding 0.17 g (21%) of intermediate 75.

Example A13 a) Preparation of Intermediate 76

A mixture of 6-methoxy-1H-indole-3-ethanamine (0.053 mol) and1,3-isobenzofurandione (0.058 mol) in toluene (130 ml) was stirred andrefluxed for 48 hours, then filtered. The filtrate was evaporated,yielding 5.4 g (32%) of intermediate 76.

b) Preparation of Intermediate 77

A solution of intermediate 76 (0.017 mol) in DMF (19 ml) was addeddropwise at room temperature to a suspension of sodium hydride (0.034mol) in DMF (11 ml) under N₂ flow. The mixture was stirred at roomtemperature for 1 hour and 30 minutes. 1-iodo-propane (0.034 mol) wasadded. The mixture was stirred at room temperature for 1 hour and 15minutes. Saturated NaCl was added. The mixture was extracted with EtOAc.The organic layer was washed with water, dried (MgSO₄), filtered, andthe solvent was evaporated, yielding 4.9 g of intermediate 77. Thisproduct was used directly in the next reaction step.

c) Preparation of Intermediate 78

A mixture of intermediate 77 (0.068 mol) and hydrazine, monohydrate(0.068 mol) in EtOH (60 ml) was stirred and refluxed for 1 hour, pouredout into water and extracted with DCM. The organic layer was separated,dried (MgSO₄), filtered, and the solvent was evaporated, yielding 3.53 gof intermediate 78. This product was used directly in the next reactionstep.

d) Preparation of Intermediate 79

Sodiumcyanoborohydride (0.024 mol) and acetic acid (0.0167 mol) wereadded at room temperature to a solution of intermediate 61 (0.0167 mol)and intermediate 78 (0.015 mol) in MeOH (380 ml) under N₂ flow. Themixture was stirred for 30 minutes, poured out into potassium carbonate10% and extracted with DCM. The organic layer was separated, dried(MgSO₄), filtered, and the solvent was evaporated. The residue (8.84 g)was purified by column chromatography over silica gel (15-40 μm)(eluent: DCM/MeOH/NH₄OH 96/4/0.2). The pure fractions were collected andthe solvent was evaporated, yielding 2.85 g (40%) of intermediate 79.

e) Preparation of Intermediate 80

A mixture of intermediate 79 (0.0028 mol), iodo-ethane (0.0056 mol) andtriethylamine (0.0085 mol) in DMF (60 ml) was stirred at 50° C. for 7hours, poured out into ice water and extracted with EtOAc. The organiclayer was washed with water, dried (MgSO₄), filtered and the solvent wasevaporated. The residue (1.8 g) was purified by column chromatographyover kromasil (5 μm) (eluent: DCM/MeOH 100/0 to 95/5). The purefractions were collected and the solvent was evaporated, yielding 1.1 g(78%) of intermediate 80.

f) Preparation of Intermediate 81

A mixture of intermediate 80 (0.0022 mol) and sodium hydroxide (0.0088mol) in EtOH (100 ml) was stirred and refluxed for 6 hours, then stirredat room temperature overnight and evaporated till dryness. The residuewas taken up in diethyl ether. The precipitate was filtered off anddried, yielding 0.965 g (88%) of intermediate 81, melting point >260° C.

g) Preparation of Intermediate 82

EDC (0.0038 mol) and HOBt (0.0038 mol) were added at room temperature toa solution of intermediate 81 (0.0019 mol) in THF (100 ml) and DCM (100ml) under N₂ flow. The mixture was stirred at room temperature for 30minutes. O-(tetrahydro-2H-pyran-2-yl)-hydroxylamine (0.0038 mol) wasadded. The mixture was stirred at room temperature for 48 hours, pouredout into water and extracted with DCM. The organic layer was separated,dried (MgSO₄), filtered and the solvent was evaporated. The residue (1.2g) was purified by column chromatography over kromasil (5 μm) (eluent:DCM/MeOH/NH₄OH 99/1/0.05 to 93/7/0.35). The pure fractions werecollected and the solvent was evaporated, yielding 0.114 g ofintermediate 82.

Example A14 a) Preparation of Intermediate 83

A mixture of intermediate 62 (0.0034 mol) and sodium hydroxide (0.0134mol) in EtOH (150 ml) was stirred at 80° C. for 3 hours, then cooled toroom temperature and evaporated till dryness. The residue was taken upin diethyl ether. The precipitate was filtered off and dried, yielding1.18 g (78%) of intermediate 83, melting point >260° C.

b) Preparation of Intermediate

EDC (0.0052 mol) and HOBt (0.0052 mol) were added at room temperature toa solution of intermediate 83 (0.0026 mol) in THF (120 ml) and DCM (120ml) under N₂ flow. The mixture was stirred at room temperature for 30minutes. O-(tetrahydro-2H-pyran-2-yl)-hydroxylamine (0.0052 mol) wasadded. The mixture was stirred at room temperature for 6 days, pouredout into ice water and extracted with DCM. The organic layer wasseparated, dried (MgSO₄), filtered and the solvent was evaporated. Theresidue (2 g) was purified by column chromatography over silica gel(15-40 μm) (eluent: DCM/MeOH/NH₄OH 96/4/0.5). The pure fractions werecollected and the solvent was evaporated. The residue (0.75 g, 55%) waspurified by column chromatography over kromasil (10 μm) (eluent:DCM/MeOH/NH₄OH 96/4/0.5). The pure fractions were collected and thesolvent was evaporated, yielding 0.625 g of intermediate 84. Thisproduct was used directly in the next reaction step.

Example A15 Preparation of Intermediate 85

4-Piperidinemethanamine (0.65 mol) and potassium carbonate (96 g) werestirred in acetonitrile (1000 ml) and then a solution of2-(methylsulfonyl)-5-pyrimidinecarboxylic acid, ethyl ester (0.37 mol)in acetonitrile (500 ml) was added dropwise at room temperature over 1hour. The reaction mixture was stirred overnight at room temperature andthe solvent was evaporated. The residue was stirred in water and themixture was extracted with DCM (2×500 ml). The organic layer wasseparated, washed with water, dried (MgSO₄), filtered off and thesolvent was evaporated. The residue was purified by columnchromatography over silica gel (eluent 1: EtOAc/Hexane 1/1; eluent 2:MeOH+small amount of NH₄OH). The product fractions were collected,stirred with potassium carbonate slurry and the mixture was extractedwith DCM. The organic layer was separated, dried (MgSO₄), filtered offand the solvent was evaporated, yielding 31 g (32%) of intermediate 85.

Example A16 a) Preparation of Intermediate 86

Sodium hydride (0.0095 mol) was added at 5° C. to a solution of5-chloro-1H-indole-3-carboxaldehyde (0.0056 mol) in THF (52 ml) under N₂flow. The mixture was stirred at 0° C. for 1 hour. 1-Iodo-propane(0.0067 mol) was added. The mixture was stirred at room temperature for2 days, poured out into water and extracted with EtOAc. The organiclayer was separated, dried (MgSO₄), filtered, and the solvent wasevaporated, yielding 1.5 g of intermediate 86. This product was useddirectly in the next reaction step.

b) Preparation of Intermediate 87

Sodiumcyanoborohydride (0.0068 mol) and acetic acid (0.0046 mol) wereadded at room temperature to a solution of intermediate 85 (0.0042 mol)and intermediate 86 (0.0051 mol) in MeOH (120 ml) under N₂ flow. Themixture was stirred and refluxed for 2 days, then cooled to roomtemperature, poured out into potassium carbonate 10% and extracted withDCM. The organic layer was separated, dried (MgSO₄), filtered, and thesolvent was evaporated. The residue (2.42 g) was purified by columnchromatography over silica gel (15-40 μm) (eluent: DCM/MeOH/NH₄OH95/5/0.2). The pure fractions were collected and the solvent wasevaporated, yielding 1.2 g (60%) of intermediate 87.

c) Preparation of Intermediate 88

A mixture of intermediate 87 (0.0025 mol) and sodium hydroxide (0.01mol) in EtOH (120 ml) was stirred and refluxed for 4 hours, thenevaporated till dryness. The residue was taken up in diethyl ether. Theprecipitate was filtered off and dried, yielding 0.845 g (72%) ofintermediate 88, melting point >260° C.

d) Preparation of Intermediate 89

EDC (0.0036 mol) and HOBt (0.0036 mol) were added at room temperature toa solution of intermediate 88 (0.0018 mol) in THF (90 ml) and DCM (90ml) under N₂ flow. The mixture was stirred for 30 minutes.O-(tetrahydro-2H-pyran-2-yl)-hydroxylamine (0.0036 mol) was added. Themixture was stirred at room temperature for 3 days, poured out into icewater and extracted with DCM. The organic layer was separated, dried(MgSO₄), filtered, and the solvent was evaporated. The residue (1.3 g)was purified by column chromatography over silica gel (15-40 μm)(eluent: DCM/MeOH/NH₄OH 92/8/0.5). The pure fractions were collected andthe solvent was evaporated, yielding 0.54 g (56%) of intermediate 89.

Example A17 a) Preparation of Intermediate 90

Methanesulfonyl chloride (0.004 mol) was added at 10° C. to a solutionof 1,2-dimethyl-1H-indole-3-ethanol (0.0026 mol) and triethyl amine(0.008 mol) in DCM (10 ml) under N₂ flow. The mixture was stirred at 10°C. for 4 hours. The solvent was evaporated till dryness, yieldingintermediate 90. This product was used directly in the next reactionstep.

b) Preparation of Intermediate 91

A mixture of intermediate 85 (0.0054 mol), intermediate 90 (0.0075 mol)and potassium carbonate (0.021 mol) in acetonitrile (150 ml) was stirredand refluxed for 2 days, then cooled to room temperature, poured outinto ice water and extracted with EtOAc. The organic layer wasseparated, dried (MgSO₄), filtered, and the solvent was evaporated. Theresidue (1.88 g) was purified by column chromatography over silica gel(15-40 μm) (eluent: DCM/MeOH/NH₄OH 97/3/0.1). The pure fractions werecollected and the solvent was evaporated, yielding 0.15 g (7%) ofintermediate 91.

c) Preparation of Intermediate

A mixture of intermediate 91 (0.0003 mol) in sodium hydroxide (0.0014mol) and EtOH (20 ml) was stirred and refluxed for one day, then cooledto room temperature and evaporated till dryness. The residue was takenup in diethyl ether. The precipitate was filtered off and dried,yielding 0.12 g (82%) of intermediate 92, melting point >260° C.

d) Preparation of Intermediate 93

EDC (0.0005 mol) and HOBt (0.0005 mol) were added at room temperature toa solution of intermediate 92 (0.0002 mol) in THF (15 ml) and DCM (15ml). The mixture was stirred for 15 minutes.O-(tetrahydro-2H-pyran-2-yl)-hydroxylamine (0.0005 mol) was added. Themixture was stirred at room temperature for 4 days, poured out into icewater and extracted with DCM. The organic layer was separated, dried(MgSO₄), filtered, and the solvent was evaporated. The residue (0.14 g)was purified by column chromatography over silica gel (10 μm) (eluent:DCM/MeOH/NH₄OH 95/5/0.3). The pure fractions were collected and thesolvent was evaporated, yielding 0.035 g (25%) of intermediate 93.

Table F-1 lists the intermediates that were prepared according to one ofthe above Examples.

TABLE F-1 (intermediates)

Interm. 109; Ex. [A1]; mp. °C.

B. Preparation of the Final Compounds Example B1 Preparation of Compound1a

TFA (2 ml) was added to a mixture of intermediate 3 (0.0006 mol) in MeOH(40 ml). The mixture was stirred at room temperature for 30 hours. Thesolvent was evaporated. The residue was crystallized from EtOAc/diethylether. The precipitate was filtered off and dried, yielding 0.31 g (86%)of compound 1a, melting point 130° C.

Alternative Synthesis for Compound 1 Preparation of Compound 1b

Hydroxylamine (50% in water, 7.5 ml) and then NaOH 1N (15 ml) were addedat 10° C. to a mixture of intermediate 1 (0.0098 mol) in MeOH (10 ml).The mixture was stirred at room temperature for 24 hours. The mixturewas acidified to pH5-6 by adding a solution of HCl 1N. The precipitatewas filtered off washed with diethylether and dried. The residue was(4.5 g) was purified by column chromatography over silica gelLiChroprep® NH2 (25-40 μm) (eluent: DCM/MeOH/H₂O 90/10/1). The purefractions were collected and the solvent was evaporated, yielding 3.1 g(80%). The HCl salt was prepared on a fraction (0.5 g) in EtOH and theprecipitate was filtered off washed with diethylether and dried yielding0.43 g of compound 1b, melting point 220° C.

Example B2 Preparation of Compound 2

TFA (0.5 ml) was added to a mixture of intermediate 7 (0.0001 mol) inMeOH (10 ml) and the mixture was stirred at room temperature for 24hours. The solvent was evaporated. The residue was crystallized fromacetonitrile/diethyl ether. The precipitate was filtered off and dried,yielding 0.036 g (50%) of compound 2, melting point 205° C.

Example B3 Preparation of Compound 3

TFA (5 ml) was added at room temperature to a mixture of intermediate 12(0.0014 mol) in MeOH (100 ml). The mixture was stirred at roomtemperature for 48 hours. The solvent was evaporated till dryness. Theresidue was crystallized from EtOAc/diethyl ether. The precipitate wasfiltered off and dried, yielding 0.545 g (74%) of compound 3, meltingpoint 121° C.

Example B4 Preparation of Compound 4

TFA (0.5 ml) was added to a mixture of intermediate 17 (0.0009 mol) inMeOH (80 ml). The mixture was stirred at room temperature for 4 days.The solvent was evaporated. The residue was crystallized from diethylether. The precipitate was filtered off and dried, yielding 0.19 g (32%)of compound 4, melting point 103° C.

Example B5 Preparation of Compound 18

A mixture of intermediate 48 (0.0002 mol) in TFA (0.75 ml) and MeOH (15ml) was stirred at room temperature for 24 hours. The solvent wasevaporated. The residue was crystallized from diethyl ether. Theprecipitate was filtered off and dried, yielding 0.071 g (59%) ofcompound 18.

Example B6 Preparation of Compound 19

A mixture of intermediate 53 (0.0003 mol) in TFA (1 ml) and MeOH (20 ml)was stirred at room temperature for 24 hours. The solvent was evaporatedtill dryness. The residue was crystallized from MeOH/CH₃CN/diethylether. The precipitate was filtered off and dried, yielding 0.133 g(80%) of compound 19, melting point 174° C.

Example B7 Preparation of Compound 20

A mixture of intermediate 57 (0.00005 mol) in TFA (0.25 ml) and MeOH (10ml) was stirred at room temperature for 24 hours. The solvent wasevaporated. The residue (0.04 g) was crystallized from CH₃CN/diethylether. The precipitate was filtered off and dried. The residue (0.04 g)was purified by column chromatography over silica gel LiChroprep® NH₂(25-40 μm) (eluent: DCM/MeOH/H₂O 80/20/2). The pure fractions werecollected and the solvent was evaporated, yielding 0.02 g (80%) ofcompound 20, melting point 90° C.

Example B8 Preparation of Compound 21

A mixture of intermediate 60 (0.0001 mol) in TFA (0.5 ml) and MeOH (10ml) was stirred at room temperature overnight. The solvent wasevaporated. The residue (0.15 g) was purified by column chromatographyover silica gel LiChroprep® NH₂ (25-40 μm) (eluent: DCM/MeOH/H₂O80/20/2). The pure fractions were collected and the solvent wasevaporated, yielding 0.064 g (78%) of compound 21, melting point 83° C.

Example B9 Preparation of Compound 22

A mixture of intermediate 65 (0.002 mol) in TFA (6 ml) and MeOH (120 ml)was stirred at room temperature for 24 hours. The solvent was evaporatedtill dryness. The residue was crystallized from CH₃CN/MeOH/diethylether. The precipitate was filtered off and dried, yielding 0.9 g (87%)of compound 22, melting point 183° C.

Example B10 Preparation of Compound 23

A mixture of intermediate 72 (0.0001 mol) in TFA (0.5 ml) and MeOH (10ml) was stirred at room temperature for 48 hours. The solvent wasevaporated. The residue was crystallized from diethyl ether. Theprecipitate was filtered off and dried, yielding 0.059 g (59%) ofcompound 23, melting point 182° C.

Example B11 Preparation of Compound 24

A mixture of intermediate 75 (0.0003 mol) in TFA (1 ml) and MeOH (20 ml)was stirred at room temperature for 24 hours. The solvent was evaporatedtill dryness. The residue was crystallized from MeOH/CH3CN/diethylether. The precipitate was filtered off and dried, yielding 0.147 g(78%) of compound 24, melting point 160° C.

Example B12 Preparation of Compound 25

A mixture of intermediate 82 (0.0009 mol) in TFA (2.5 ml) and MeOH (56ml) was stirred at room temperature for 24 hours. The solvent wasevaporated till dryness. The residue was purified by columnchromatography over silica gel (25-40 μm) (eluent: DCM/MeOH/H₂O90/10/1). The pure fractions were collected and the solvent wasevaporated. The residue was crystallized from DIPE. The precipitate wasfiltered off and dried, yielding 0.286 g (53%) of compound 25, meltingpoint 80° C.

Example B13 Preparation of Compound 26

A mixture of intermediate 84 (0.0012 mol) in TFA (3 ml) and MeOH (60 ml)was stirred at room temperature for 24 hours. The solvent was evaporatedtill dryness. The residue was crystallized from DCM/MeOH. Theprecipitate was filtered off, washed with diethyl ether and dried,yielding 0.322 g (50%) of compound 26, melting point 188° C.

Example B14 Preparation of Compound 27

A mixture of intermediate 89 (0.001 mol) in TFA (2.5 ml) and MeOH (50ml) was stirred at room temperature for 24 hours, then evaporated tilldryness. The residue was crystallized from MeOH/CH₃CN/diethyl ether. Theprecipitate was filtered off, washed with water and dried, yielding 0.33g (55%) of compound 27, melting point 171° C.

Example B15 Preparation of Compound 28

A mixture of intermediate 93 (0.00007 mol) in TFA (0.2 ml) and MeOH (4ml) was stirred at room temperature for 3 days, then evaporated tilldryness, yielding 0.041 g (100%) of compound 28, melting point 80° C.

Table F-2 lists the compounds that were prepared according to one of theabove Examples. The following abbreviations were used in the tables:.C₂HF₃O₂ stands for the trifluoroacetate salt, mp. stands for meltingpoint.

TABLE F-2 (final compounds)

C. Pharmacological Example

The in vitro assay for inhibition of histone deacetylase (see exampleC.1) measures the inhibition of HDAC enzymatic activity obtained withthe compounds of formula (I).

Cellular activity of the compounds of formula (I) was determined onA2780 tumour cells using a colorimetric assay for cell toxicity orsurvival (Mosmann Tim, Journal of Immunological Methods 65: 55-63,1983)(see example C.2).

The solubility of a compound measures the ability of a compound to stayin solution. In a first method the ability of a compound to stay inaqueous solution upon dilution (see example C.3.a) is measured.DMSO-stock solutions are diluted with a single aqueous buffer solvent in3 consecutive steps. For every dilution turbidity is measured with anephelometer.

In a second method the solubility of a compound at different pH's can bemeasured with the use of a Chemiluminescent Nitrogen Detector (seeexample C.3.b).

A drug's permeability expresses its ability to move from one medium intoor through another. Specifically its ability to move through theintestinal membrane into the blood stream and/or from the blood streaminto the target. Permeability (see example C.4) can be measured throughthe formation of a filter-immobilized artificial membrane phospholipidbilayer. In the filter-immobilized artificial membrane assay, a“sandwich” is formed with a 96-well microtitre plate and a 96-wellfilter plate, such that each composite well is divided into two chamberswith a donor solution at the bottom and an acceptor solution at the top,separated by a 125 μm micro-filter disc (0.45 μm pores), coated with 2%(wt/v) dodecane solution of dioleoylphosphatidyl-choline, underconditions that multi-lamellar bilayers form inside the filter channelswhen the system contacts an aqueous buffer solution. The permeability ofcompounds through this artificial membrane is measured in cm/s. Thepurpose is to look for the permeation of the drugs through a parallelartificial membrane at 2 different pH's: 4.0 and 7.4. Compound detectionis done with UV-spectrometry at optimal wavelength between 250 and 500nm.

Metabolism of drugs means that a lipid-soluble xenobiotic or endobioticcompound is enzymatically transformed into (a) polar, water-soluble, andexcretable metabolite(s). The major organ for drug metabolism is theliver. The metabolic products are often less active than the parent drugor inactive. However, some metabolites may have enhanced activity ortoxic effects. Thus drug metabolism may include both “detoxication” and“toxication” processes. One of the major enzyme systems that determinethe organism's capability of dealing with drugs and chemicals isrepresented by the cytochrome P450 monooxygenases, which are NADPHdependent enzymes. Metabolic stability of compounds can be determined invitro with the use of subcellular human tissue (see example C.5.a.).Here metabolic stability of the compounds is expressed as % of drugmetabolised after 15 minutes incubation of these compounds withmicrosomes. Quantitation of the compounds was determined by LC-MSanalysis. Metabolic stability of compounds can also be determined bycalculating the half live of compounds in rat hepatocyte cells (seeexample C.5.b.).

It has been shown that a wide variety of anti-tumoral agents activatethe p21 protein, including DNA damaging agents and histone deacetylaseinhibitors. DNA damaging agents activate the p21 gene through the tumoursuppressor p53, while histone deacetylase inhibitors transcriptionallyactivates the p21 gene via the transcription factor Sp1. Thus, DNAdamaging agents activate the p21 promoter through the p53 responsiveelement while histone deacetylase inhibitors activate the p21 promoterthrough sp1 sites (located at the −60 bp to +40 bp region relative tothe TATA box) both leading to increased expression of the p21 protein.When the p21 promoter in a cells consists of a p21 1300 bp promoterfragment that does not comprise the p53 responsive elements it isaccordingly non-responsive to DNA damaging agents. The capacity ofcompounds to induce p21 can be evaluated in several ways. A first methodis to treat tumour cells with the compound of interest and after lysisof the cells detects p21 induction with the p21 enzyme linkedimmunosorbent assay (WAF1 ELISA of Oncogene). The p21 assay is a“sandwich” enzyme immunoassay employing both mouse monoclonal and rabbitpolyclonal antibodies. A rabbit polyclonal antibody, specific for thehuman p21 protein, has been immobilized onto the surface of the plasticwells provided in the kit. Any p21 present in the sample to be assayedwill bind to the capture antibody. The biotinylated detector monoclonalantibody also recognizes human p21 protein, and will bind to any p21,which has been retained by the capture antibody. The detector antibody,in turn, is bound by horseradish peroxidase-conjugated streptavidin. Thehorseradish peroxidase catalyses the conversion of the chromogenicsubstrate tetra-methylbenzidine from a colorless solution to a bluesolution (or yellow after the addition of stopping reagent), theintensity of which is proportional to the amount of p21 protein bound tothe plate. The colored reaction product is quantified using aspectrophotometer. Quantitation is achieved by the construction of astandard curve using known concentrations of p21 (provided lyophilised).This assay can measures p21 induction as the consequence of DNA damageor as the consequence of histone deacetylase inhibition (see exampleC.6.a.).

Another method tests the capacity of compounds to induce p21 as theconsequence of HDAC inhibition at the cellular level. The cells can bestably transfected with an expression vector containing a p21 1300 bppromoter fragment that does not comprise the p53 responsive elements andwherein an increase of a reporter gene expression, compared to thecontrol levels, identifies the compound as having p21 inductioncapacity. The reporter gene is a fluorescent protein and the expressionof the reporter gene is measured as the amount of fluorescent lightemitted (see example C.6.b.). The last method is an in vivo methodwherein mice are used for screening the pharmaceutical activity of acompound. The above described stably transformed tumour cells can beadministered to mice in an amount sufficient to effect production of atumour. After the tumour cells had sufficient time to form a tumour, apotentially active compound can be administered to the animals and theeffect of said compound on the tumour cells is evaluated by measuringthe expression of the reporter gene. Incubation with pharmaceuticalactive compounds will result in an increase of reporter gene expressioncompared to the control levels (see example C.6.c.)

Specific HDAC inhibitors should not inhibit other enzymes like theabundant CYP P450 proteins. The CYP P450 (E. coli expressed) proteins3A4, 2D6 en 2C9 convert their specific substrates into a fluorescentmolecule. The CYP3A4 protein converts 7-benzyloxy-trifluoromethylcoumarin (BFC) into 7-hydroxy-trifluoromethyl coumarin. The CYP2D6protein converts3-[2-(N,N-diethyl-N-methylamino)ethyl]-7-methoxy-4-methylcoumarin (AMMC)into 3-[2-(N,N-diethylamino)ethyl]-7-hydroxy-4-methylcoumarinhydrochloride and the CYP2C9 protein converts7-Methoxy-4-trifluoromethyl coumarin (MFC) into7-hydroxy-trifluoromethyl coumarin. Compounds inhibiting the enzymaticreaction will result in a decrease of fluoresent signal (see exampleC.7).

Example C.1 In Vitro Assay for Inhibition of Histone Deacetylase ExampleC.1.a: In Vitro Assay with [³H]-Labelled Substrate

HeLa nuclear extracts (supplier: Biomol) were incubated at 60 μg/ml with75 μM of substrate. As a substrate for measuring HDAC activity asynthetic peptide, i.e. the amino acids 14-21 of histone H4, was used.The substrate is biotinylated at the NH₂-terminal part with a6-aminohexanoic acid spacer, and is protected at the COOH-terminal partby an amide group and specifically [³H]acetylated at lysine 16. Thesubstrate,biotin-(6-aminohexanoic)Gly-Ala-([³H]-acetyl-Lys-Arg-His-Arg-Lys-Val-NH₂),was added in a buffer containing 25 mM Hepes, 1 M sucrose, 0.1 mg/ml BSAand 0.01% Triton X-100 at pH 7.4. After 30 min the deacetylationreaction was terminated by the addition of HCl and acetic acid. (finalconcentration 0.035 mM and 3.8 mM respectively). After stopping thereaction, the free ³H-acetate was extracted with ethylacetate. Aftermixing and centrifugation, the radioactivity in an aliquot of the upper(organic) phase was counted in a β-counter.

For each experiment, controls (containing HeLa nuclear extract and DMSOwithout compound), a blank incubation (containing DMSO but no HeLanuclear extract or compound) and samples (containing compound dissolvedin DMSO and HeLa nuclear extract) were run in parallel. In firstinstance, compounds were tested at a concentration of 10⁻⁵M. When thecompounds showed activity at 10⁻⁵M, a concentration-response curve wasmade wherein the compounds were tested at concentrations between 10⁻⁵Mand 10⁻¹²M. In each test the blank value was substracted from both thecontrol and the sample values. The control sample represented 100% ofsubstrate deactylation. For each sample the radioactivity was expressedas a percentage of the mean value of the controls. When appropriateIC₅₀-values (concentration of the drug, needed to reduce the amount ofmetabolites to 50% of the control) were computed using probit analysisfor graded data. Herein the effects of test compounds are expressed aspIC₅₀ (the negative log value of the IC₅₀-value) (see Table F-3).

Example C.1.b: In Vitro Assay with Fluorescent-Labelled Substrate

The HDAC Fluorescent Activity Assay/Drug Discovery Kit of Biomol (cat.No: AK-500-0001) was used. The HDAC Fluorescent Activity Assay is basedon the Fluor de Lys (Fluorogenic Histone deAcetylase Lysyl) substrateand developer combination. The Fluor de Lys substrate, comprises anacetylated lysine side chain. Deacetylation of the substrate sensitizesthe substrate so that, in the second step, treatment with the Fluor deLys developer produces a fluorophore.

HeLa nuclear extracts (supplier: Biomol) were incubated at 60 μg/ml with75 μM of substrate. The Fluor de Lys substrate was added in a buffercontaining 25 mM Tris, 137 mM NaCl, 2.7 mM KCl and 1 mM MgCl₂.6H₂O at pH7.4. After 30 min, 1 volume of the developer was added. The fluorophorewas excited with 355 nm light and the emitted light (450 nm) was bedetected on a fluorometric plate reader.

For each experiment, controls (containing HeLa nuclear extract andbuffer), a blank incubation (containing buffer but no HeLa nuclearextract) and samples (containing compound dissolved in DMSO and furtherdiluted in buffer and HeLa nuclear extract) were run in parallel. Infirst instance, compounds were tested at a concentration of 10⁻⁵M. Whenthe compounds showed activity at 10⁻⁵M, a concentration-response curvewas made wherein the compounds were tested at concentrations between10⁻⁵M and 10⁻⁹M. All sample were tested 4 times. In each test the blankvalue was substracted from both the control and the sample values. Thecontrol sample represented 100% of substrate deactylation. For eachsample the fluorescence was expressed as a percentage of the mean valueof the controls. When appropriate IC₅₀-values (concentration of thedrug, needed to reduce the amount of metabolites to 50% of the control)were computed using probit analysis for graded data. Herein the effectsof test compounds are expressed as pIC₅₀ (the negative log value of theIC₅₀-value) (see Table F-3).

Example C.2 Determination of Antiproliferative Activity on A2780 Cells

All compounds tested were dissolved in DMSO and further dilutions weremade in culture medium. Final DMSO concentrations never exceeded 0.1%(v/v) in cell proliferation assays. Controls contained A2780 cells andDMSO without compound and blanks contained DMSO but no cells. MTT wasdissolved at 5 mg/ml in PBS. A glycine buffer comprised of 0.1 M glycineand 0.1 M NaCl buffered to pH 10.5 with NaOH (1 N) was prepared (allreagents were from Merck).

The human A2780 ovarian carcinoma cells (a kind gift from Dr. T. C.Hamilton [Fox Chase Cancer Centre, Pa., USA]) were cultured in RPMI 1640medium supplemented with 2 mM L-glutamine, 50 μg/ml gentamicin and 10%fetal calf serum. Cells were routinely kept as monolayer cultures at 37°C. in a humidified 5% CO₂ atmosphere. Cells were passaged once a weekusing a trypsin/EDTA solution at a split ratio of 1:40. All media andsupplements were obtained from Life Technologies. Cells were free ofmycoplasma contamination as determined using the Gen-Probe MycoplasmaTissue Culture kit (supplier: BioMérieux).

Cells were seeded in NUNC™ 96-well culture plates (Supplier: LifeTechnologies) and allowed to adhere to the plastic overnight. Densitiesused for plating were 1500 cells per well in a total volume of 200 μlmedium. After cell adhesion to the plates, medium was changed and drugsand/or solvents were added to a final volume of 200 μl. Following fourdays of incubation, medium was replaced by 200 μl fresh medium and celldensity and viability was assessed using an MTT-based assay. To eachwell, 25 μl MTT solution was added and the cells were further incubatedfor 2 hours at 37° C. The medium was then carefully aspirated and theblue MTT-formazan product was solubilized by addition of 25 μl glycinebuffer followed by 100 μl of DMSO. The microtest plates were shaken for10 min on a microplate shaker and the absorbance at 540 nm was measuredusing an Emax 96-well spectrophotometer (Supplier: Sopachem). Within anexperiment, the results for each experimental condition are the mean of3 replicate wells. For initial screening purposes, compounds were testedat a single fixed concentration of 10⁻⁶ M. For active compounds, theexperiments were repeated to establish full concentration-responsecurves. For each experiment, controls (containing no drug) and a blankincubation (containing no cells or drugs) were run in parallel. Theblank value was subtracted from all control and sample values. For eachsample, the mean value for cell growth (in absorbance units) wasexpressed as a percentage of the mean value for cell growth of thecontrol. When appropriate, IC₅₀-values (concentration of the drug,needed to reduce cell growth to 50% of the control) were computed usingprobit analysis for graded data (Finney, D. J., Probit Analyses, 2^(nd)Ed. Chapter 10, Graded Responses, Cambridge University Press, Cambridge1962). Herein the effects of test compounds are expressed as pIC₅₀ (thenegative log value of the IC₅₀-value)(see Table F-3).

Example C.3 Solubility/Stability C.3.1. Solubility in Aqueas Media

In the first dilution step, 10 μl of a concentrated stock-solution ofthe active compound, solubilized in DMSO (5 mM), was added to 100 μlphosphate citrate buffer pH 7.4 and mixed. In the second dilution step,an aliquot (20 μl) of the first dilution step was further dispensed in100 μl phosphate citrate buffer pH 7.4 and mixed. Finally, in the thirddilution step, a sample (20 μl) of the second dilution step was furtherdiluted in 100 μl phosphate citrate buffer pH 7.4 and mixed. Alldilutions were performed in 96-well plates Immediately after the lastdilution step the turbidity of the three consecutive dilution steps weremeasured with a nephelometer. Dilution was done in triplicate for eachcompound to exclude occasional errors. Based on the turbiditymeasurements a ranking is performed into 3 classes. Compounds with highsolubility obtained a score of 3 and for this compounds the firstdilution is clear. Compounds with medium solubility obtained a score of2. For these compounds the first dilution is unclear and the seconddilution is clear. Compounds with low solubility obtained a score of 1and for these compounds both the first and the second dilution areunclear (see Table F-3).

C.3.b. Solubility/Stability at Different pH's

The solubility of a compound, at different pH's, can also be measuredwith the use of a chemiluminescent nitrogen detector. (see Table F-3).

Example C.4 Parallel Artificial Membrane Permeability Analysis

The stock samples (aliquots of 10 μl of a stock solution of 5 mM in 100%DMSO) were diluted in a deep-well or Pre-mix plate containing 2 ml of anaqueous buffer system pH 4 or pH 7.4 (PSR4 System Solution Concentrate(pION)).

Before samples were added to the reference plate, 150 μl of buffer wasadded to wells and a blank UV-measurement was performed. Thereafter thebuffer was discarded and the plate was used as reference plate. Allmeasurements were done in UV-resistant plates (supplier: Costar orGreiner).

After the blank measurement of the reference plate, 150 μl of thediluted samples was added to the reference plate and 200 μl of thediluted samples was added to donorplate 1. An acceptor filter plate 1(supplier: Millipore, type: MAIP N45) was coated with 4 μl of theartificial membrane-forming solution(1,2-Dioleoyl-sn-Glycer-3-Phosphocholine in Dodecane containing 0.1%2,6-Di-tert-butyl-4-methylphenol and placed on top of donor plate 1 toform a “sandwich”. Buffer (200 μl) was dispensed into the acceptor wellson the top. The sandwich was covered with a lid and stored for 18 h atroom temperature in the dark.

A blank measurement of acceptor plate 2 was performed through theaddition of 150 μl of buffer to the wells, followed by anUV-measurement. After the blank measurement of acceptor plate 2 thebuffer was discarded and 150 μl of acceptor solution was transferredfrom the acceptor filter plate 1 to the acceptor plate 2. Then theacceptor filter plate 1 was removed form the sandwich. After the blankmeasurement of donor plate 2 (see above), 150 μl of the donor solutionwas transferred from donor plate 1 to donor plate 2. The UV spectra ofthe donor plate 2, acceptor plate 2 and reference plate wells werescanned (with a SpectraMAX 190). All the spectra were processed tocalculate permeability with the PSR4p Command Software. All compoundswere measured in triplo. Carbamazepine, griseofulvin, acycloguanisine,atenolol, furosemide, and chlorothiazide were used as standards in eachexperiment. Compounds were ranked in 3 categories as having a lowpermeability (mean effect <0.5×10⁻⁶ CM/S; score 1), a mediumpermeability (1×10⁻⁶ cm/s>mean effect≧0.5×10⁻⁶ cm/s; score 2) or a highpermeability (≧1×10⁻⁶ cm/s; score 3).

Example C.5 Metabolic Stability Example C.5.a

Sub-cellular tissue preparations were made according to Gorrod et al.(Xenobiotica 5: 453-462, 1975) by centrifugal separation aftermechanical homogenization of tissue. Liver tissue was rinsed in ice-cold0.1 M Tris-HCl (pH 7.4) buffer to wash excess blood. Tissue was thenblotted dry, weighed and chopped coarsely using surgical scissors. Thetissue pieces were homogenized in 3 volumes of ice-cold 0.1 M phosphatebuffer (pH 7.4) using either a Potter-S (Braun, Italy) equipped with aTeflon pestle or a Sorvall Omni-Mix homogeniser, for 7×10 sec. In bothcases, the vessel was kept in/on ice during the homogenization process.

Tissue homogenates were centrifuged at 9000×g for 20 minutes at 4° C.using a Sorvall centrifuge or Beckman Ultracentrifuge. The resultingsupernatant was stored at −80° C. and is designated ‘S9’.

The S9 fraction can be further centrifuged at 100.000× g for 60 minutes(4° C.) using a Beckman ultracentrifuge. The resulting supernatant wascarefully aspirated, aliquoted and designated ‘cytosol’. The pellet wasre-suspended in 0.1 M phosphate buffer (pH 7.4) in a final volume of 1ml per 0.5 g original tissue weight and designated ‘microsomes’.

All sub-cellular fractions were aliquoted, immediately frozen in liquidnitrogen and stored at −80° C. until use.

For the samples to be tested, the incubation mixture contained PBS(0.1M), compound (5 μM), microsomes (1 mg/ml) and a NADPH-generatingsystem (0.8 mM glucose-6-phosphate, 0.8 mM magnesium chloride and 0.8Units of glucose-6-phosphate dehydrogenase). Control samples containedthe same material but the microsomes were replaced by heat inactivated(10 min at 95 degrees Celsius) microsomes. Recovery of the compounds inthe control samples was always 100%.

The mixtures were preincubated for 5 min at 37 degrees Celsius. Thereaction was started at timepoint zero (t=0) by addition of 0.8 mM NADPand the samples were incubated for 15 min (t=15). The reaction wasterminated by the addition of 2 volumes of DMSO. Then the samples werecentrifuged for 10 min at 900×g and the supernatants were stored at roomtemperature for no longer as 24 h before analysis. All incubations wereperformed in duplo. Analysis of the supernatants was performed withLC-MS analysis. Elution of the samples was performed on a Xterra MS C18(50×4.6 mm, 5 μm, Waters, US). An Alliance 2790 (Supplier: Waters, US)HPLC system was used. Elution was with buffer A (25 mM ammoniumacetate(pH 5.2) in H₂O/acetonitrile (95/5)), solvent B being acetonitrile andsolvent C methanol at a flow rate of 2.4 ml/min. The gradient employedwas increasing the organic phase concentration from 0% over 50% B and50% C in 5 min up to 100% B in 1 min in a linear fashion and organicphase concentration was kept stationary for an additional 1.5 min. Totalinjection volume of the samples was 25 μl.

A Quattro (supplier: Micromass, Manchester, UK) triple quadrupole massspectrometer fitted with and ESI source was used as detector. The sourceand the desolvation temperature were set at 120 and 350° C. respectivelyand nitrogen was used as nebuliser and drying gas. Data were acquired inpositive scan mode (single ion reaction). Cone voltage was set at 10 Vand the dwell time was 1 sec.

Metabolic stability was expressed as % metabolism of the compound after15 min of incubation in the presence of active microsomes

$\left( {E({act})} \right)\left( {{\%\mspace{14mu}{metabolism}} = {{100\%} - {\left( {\left( \frac{{{Total}\mspace{14mu}{Ion}\mspace{14mu}{{Current}({TIC})}{of}\mspace{14mu}{E({act})}{at}\mspace{14mu} t} = 15}{{{TIC}\mspace{14mu}{of}\mspace{14mu}{E({act})}{at}\mspace{14mu} t} = 0} \right) \times 100} \right).}}} \right.$Compounds that had a percentage metabolism less than 20% were defined ashighly metabolic stable. Compound that had a metabolism between 20 and70% were defined as intermediately stable and compounds that showed apercentage metabolism higher than 70 were defined as low metabolicstable. Three reference compounds were always included whenever ametabolic stability screening was performed. Verapamil was included as acompound with low metabolic stability (% metabolism=73%). Cisapride wasincluded as a compound with medium metabolic stability (% metabolism45%) and propanol was included as a compound with intermediate to highmetabolic stability (25% metabolism). These reference compounds wereused to validate the metabolic stability assay.

C.5.b: Metabolic Stability with Rat Hepatocytes Cell Culture

Rat hepatocytes were isolated from male Sprague Dowley rats. Thecompounds were dissolved to a 5 mM stock solution in 100% DMSO andincubated at a final concentration of 5 μM for 0, 15, 30, 60 and 120 minwith rat hepatocyte cell cultures (0.5 million viable cells/0.5 ml)using 24-well plates.

Samples were prepared for LC-MS by addition of two volumes of DMSO. Thesamples were thoroughly shaken and subsequently centrifuged at 900 g for10 min (room temperature). All experiments were performed in triplicate.Of the resulting supernatant 50 μl was analysed by LC-MS.

For LC-MS, elution of samples was performed on a Hypersil BDS C18 column(50×4.6 mm, 5 μm, Thermohypersil, UK). The HPLC system comprised aSurveyor delivery system (Surveyor Inc., San Jose, US) equipped with aSurveyor autosampler device. Elution was with buffer A (10 mMammoniumacetate (pH 6.9) in H₂O/Acetonitrile (95:5)) and solvent B(acetonitrile) at a flow rate of 1.2 ml/min. The gradient employed was0.5 min solvent A as start condition followed by increasing the organicphase concentration from 0% B till 95% B over 2 min in a linear fashion.This phase was kept stationary for a further 2 min and reduced again to0% B within 0.5 min.

Total injection volume of samples was 50 μL. Column oven temperature waskept at 40° C. The LC flow was splitted for MS detection and 0.1 ml letinto the source. An triple quadrupol mass spectrometer TSQ Quantum(Thermofinnigan, LaJolla, USA) mass spectrometer fitted with an ESIsource was used for detection. Source voltage was set at 3800 volt, thecapillary temperature at 300° C. The mass spectrometer was operated inpositive ion mode in SIM adjusted to the mass of M+H with a scan widthof 1 Da for quantification purposes. Instrument control, dataacquisition and processing were performed using the Xcalibur software(ThermoFinnigan, San Jose, Calif., U.S.A). The metabolic stability ofcompounds in rat hepatocytes was expressed as in vitro half-lives.

As reference, compound R306465 (WO03/76422) was used (in vitrohalf-live: 8 min) Compound 1 and compound 5 were tested and had an invitro half-live of 81 min. and 60 min. respectively.

Example C.6 p21 Induction Capacity Example C.6.a: p21 Enzyme LinkedImmunosorbent Assay

The following protocol has been applied to determine the p21 proteinexpression level in human A2780 ovarian carcinoma cells. The A2780 cells(20000 cells/180 μl) were seeded in 96 microwell plates in RPMI 1640medium supplemented with 2 mM L-glutamine, 50 μg/ml gentamicin and 10%fetal calf serum. 24 hours before the lysis of the cells, compounds wereadded at final concentrations of 10⁻⁵, 10⁻⁶, 10⁻⁷ and 10⁻⁸ M. Allcompounds tested were dissolved in DMSO and further dilutions were madein culture medium. 24 hours after the addition of the compound, thesupernatants were removed from the cells. Cells were washed with 200 μlice-cold PBS. The wells were aspirated and 30 μl of lysisbuffer (50 mMTris.HCl (pH 7.6), 150 mM NaCl, 1% Nonidet p40 and 10% glycerol) wasadded. The plates were incubated overnight at −70° C.

The appropriate number of microtiter wells were removed from the foilpouch and placed into an empty well holder. A working solution (1×) ofthe Wash Buffer (20× plate wash concentrate: 100 ml 20-fold concentratedsolution of PBS and surfactant. Contains 2% chloroacetamide) wasprepared. The lyophilised p21WAF standard was reconstituted withdistilled H₂O and further diluted with sample diluent (provided in thekit)

The samples were prepared by diluting them 1:4 in sample diluent. Thesamples (100 μl) and the p21WAF1 standards (100 μl) were pipetted intothe appropriate wells and incubated at room temperature for 2 hours. Thewells were washed 3 times with 1× wash buffer and then 100 μl ofdetector antibody reagent (a solution of biotinylated monoclonal p21WAF1antibody) was pipetted into each well. The wells were incubated at roomtemperature for 1 hour and then washed three times with 1× wash buffer.The 400× conjugate (peroxidase streptavidine conjugate: 400-foldconcentrated solution) was diluted and 100 μl of the 1× solution wasadded to the wells. The wells were incubated at room temperature for 30min and then washed 3 times with 1× wash buffer and 1 time withdistilled H₂O. Substrate solution (chromogenic substrate)(100 μl) wasadded to the wells and the wells were incubated for 30 minutes in thedark at room temperature. Stop solution was added to each well in thesame order as the previously added substrate solution. The absorbance ineach well was measured using a spectrophotometric plate reader at dualwavelengths of 450/595 nm.

For each experiment, controls (containing no drug) and a blankincubation (containing no cells or drugs) were run in parallel. Theblank value was substracted from all control and sample values. For eachsample, the value for p21WAF1 induction (in absorbance units) wasexpressed as the percentage of the value for p21WAF1 present in thecontrol. Percentage induction higher than 130% was defined assignificant induction. Nine compounds were tested and all showedsignificant induction at 10-⁶M.

Example C.6.b: Cellular Method

A2780 cells (ATCC) were cultivated in RPMI 1640 medium supplemented with10% FCS, 2 mM L-glutamine and gentamycine at 37° C. in a humidifiedincubator with 5% CO₂. All cell culture solutions are provided byGibco-BRL (Gaithersburg, Md.). Other materials are provided by Nunc.

Genomic DNA was extracted from proliferating A2780 cells and used astemplate for nested PCR isolation of the p21 promoter. The firstamplification was performed for 20 cycles at an annealing temperature of55° C. using the oligonucleotide pair GAGGGCGCGGTGCTTGG andTGCCGCCGCTCTCTCACC with the genomic DNA as template. The resulting 4.5kb fragment containing the −4551 to +88 fragment relative to the TATAbox was re-amplified with the oligonucleotidesTCGGGTACCGAGGGCGCGGTGCTTGG and ATACTCGAGTGCCGCCGCTCTCTCACC for 20 cycleswith annealing at 88° C. resulting in a 4.5 kb fragment and subsequentlywith the oligonucleotide pair TCGGGTACCGGTAGATGGGAGCGGATAGACACATC andATACTCGAGTGCCGCCGCTCTCTCACC for 20 cycles with annealing at 88° C.resulting in a 1.3 kb fragment containing the −1300 to +88 fragmentrelative to the TATA box. The restriction sites XhoI and KpnI present inthe oligonucleotides (underlined sequence) were used for subcloning.

The luciferase reporter was removed from the pGL3-basic and replaced bythe ZsGreen reporter (from the pZsGreenl-N1 plasmid) at KpnI and XbaIrestriction sites. pGL3-basic-ZsGreen-1300 was constructed via insertionof the above mentioned 1.3 kb fragment of the human p21 promoter regioninto pGL3-basic-ZsGreen at the XhoI and KpnI sites. All restrictionenzymes are provided by Boehringer Manheim (Germany). A2780 cells wereplated into a 6-well plate at a density of 2×10⁵ cells, incubated for 24hours, and transfected with 2 ug of pGL3-basic-ZsGreen-1300 and 0.2 ugof pSV2neo vector by using Lipofectamine 2000 (Invitrogen, Brussels,Belgium) as described by manufacturer. The transfected cells wereselected for 10 days with G418 (Gibco-BRL, Gaithersburg, Md.) and singlecell suspensions were grown. After three weeks, single clones wereobtained.

The A2780 selected clones were expanded and seeded at 10000 cells perwell into 96-well plates. 24 hours after seeding, the cells were treatedfor an additional 24 hours with compounds (affecting sp1 sites in theproximal p21 promoter region). Subsequently, cells were fixed with 4%PFA for 30′ and counterstained with Hoechst dye. The p21 promoteractivation leading to ZsGreen production and thus fluorescence, wasmonitored by the Ascent Fluoroskan (Thermo Labsystems, Brussels,Belgium).

For each experiment, controls (containing no drug) and a blankincubation (containing no cells or drugs) were run in parallel. Theblank value was substracted from all control and sample values. For eachsample, the value for p21 induction was expressed as the percentage ofthe value for p21 present in the control. Percentage induction higherthan 130% was defined as significant induction.

Seventy one compounds were tested and all showed significant inductionat 10⁻⁶M.

Example C.6.c: In Vivo Method

A selected clone was injected subcutaneous (10⁷ cells/200 μl) into theflank of nude mice and a calliper measurable tumour was obtained after12 days. From day 12 on, animals were dosed, orally or intraveinally,daily during 6 days with solvent and 20-40 mpk compound (4-10 animalseach). Tumours were evaluated for fluorescence by the in-house developedAutomated Whole Body Imaging System (Fluorescent stereomicroscope typeOlympus® SZX12 equipped with a GFP filter and coupled to a CCD cameratype JAI® CV-M90 controlled by a software package based on the IMAQVision Software from National Instruments®). As reference, compoundR306465 (WO03/76422) was used. Compounds were ranked as inactive (nofluorescence measurable), weaker, identical or better than R306465.Compound 1 was tested and was better than R306465.

Example C.7 P450 Inhibiting Capacity

All compounds tested were dissolved in DMSO (5 mM) and a furtherdilution to 5 10⁻⁴ M was made in acetonitrile. Further dilutions weremade in assay buffer (0.1M NaK phosphate buffer pH 7.4) and the finalsolvent concentration was never higher than 2%.

The assay for the CYP3A4 protein comprises per well 15 pmol P450/mgprotein (in 0.01M NaKphosphate buffer+1.15% KCl), an NADPH generatingsystem (3.3 mM Glucose-6-phosphate, 0.4 U/ml Glucose-6-phosphatedehydrogenase, 1.3 mM NADP and 3.3 mM MgCl₂.6H₂O in assay buffer) andcompound in a total assay volume of 100 μl. After a 5 min pre-incubationat 37° C. the enzymatic reaction was started with the addition of 150 μMof the fluoresent probe substrate BFC in assay buffer. After anincubation of 30 minutes at room temperature the reaction was terminatedafter addition of 2 volumes of acetonitrile. Fluorescent determinationswere carried out at an excitation wavelength of 405 nm and an emissionwavelength of 535 nm. Ketoconazole (IC₅₀-value=3×10⁻⁸M) was included asreference compound in this experiment. The assay for the CYP2D6 proteincomprises per well 6 pmol P450/mg protein (in 0.01M NaKphosphatebuffer+1.15% KCl), an NADPH generating system (0.41 mMGlucose-6-phosphate, 0.4 U/ml Glucose-6-phosphate dehydrogenase, 0.0082mM NADP and 0.41 mM MgCl₂.6H₂O in assay buffer) and compound in a totalassay volume of 100 μl. After a 5 min pre-incubation at 37° C. theenzymatic reaction was started with the addition of 3 μM of thefluoresent probe substrate AMMC in assay buffer. After an incubation of45 minutes at room temperature the reaction was terminated afteraddition of 2 volumes of acetonitrile. Fluorescent determinations werecarried out at an excitation wavelength of 405 nm and an emissionwavelength of 460 nm. Quinidine (IC₅₀-value <5×10⁻⁸M) was included asreference compound in this experiment.

The assay for the CYP2C9 protein comprises per well 15 pmol P450/mgprotein (in 0.01M NaKphosphate buffer+1.15% KCl), an NADPH generatingsystem (3.3 mM Glucose-6-phosphate, 0.4 U/ml Glucose-6-phosphatedehydrogenase, 1.3 mM NADP and 3.3 mM MgCl₂.6H₂O in assay buffer) andcompound in a total assay volume of 100 μl. After a 5 min pre-incubationat 37° C. the enzymatic reaction was started with the addition of 200 μMof the fluoresent probe substrate MFC in assay buffer. After anincubation of 30 minutes at room temperature the reaction was terminatedafter addition of 2 volumes of acetonitrile. Fluorescent determinationswere carried out at an excitation wavelength of 405 nm and an emissionwavelength of 535 nm.

Sulfaphenazole (IC₅₀-value=6.8×10⁻⁷M) was included as reference compoundin this experiment.

For initial screening purposes, compounds were tested at a single fixedconcentration of 1×10⁻⁵ M. For active compounds, the experiments wererepeated to establish full concentration-response curves. For eachexperiment, controls (containing no drug) and a blank incubation(containing no enzyme or drugs) were run in parallel. All compounds wereassayed in quadruplicate. The blank value was subtracted from allcontrol and sample values. For each sample, the mean value of P450activity of the sample (in relative fluorescence units) was expressed asa percentage of the mean value of P450 activity of the control.Percentage inhibition was expressed as 100% minus the mean value of P450activity of the sample. When appropriate, IC₅₀-values (concentration ofthe drug, needed to reduce P450 activity to 50% of the control) werecalculated.

TABLE F-3 lists the results of the compounds that were tested accordingto example C.1.a., C.1.b, C.2, C.3.a. and C.3.b. Enzymatic EnzymaticCellular Solubility activity activity activity C.3.b. Compound pIC50pIC50 pIC50 Solubility pH = 2.3 number C.1.a. C.1.b C.2 C.3.a. (mg/ml) 5 8.6 6.5 2.0  1a 8.8 9.2 8.2 3 1.4  6 8.2 6.3  7 8.5 6.1  3 8.5 6.7  48.7 7.0 3 17 7.8 6.8  8 8.3 7.1 2.4  9 8.3 7.1 10 8.1 7.5 3 3.7 16 8.67.1 3 11 8.2 7.5 3 2.8  2 8.4 7.5 3 15 8.5 7.5 14 8.4 7.4 1.7 13 6.0 7.53 12 8.2 7.1 3 35 8.2 6.7 68 >9.0 7.2 2.5 63 >9.0 7.5 67 8.3 7.5 1.9 767.8 6.7 72 7.9 6.8 80 7.8 7.1 34 8.4 7.5 33 10.0 7.5 1.6 32 8.0 8.4 1.521 8.5 7.6 1.9 49 8.7 7.5 3.3 53 10.0 8.0 20 9.5 8.1 2.7 64 8.9 7.8 629.5 8.0 28 8.1 7.1 61 8.9 7.6 60 8.7 6.9 3.1 59 9.0 7.7 58 10.0 5.8 198.0 7.1 24 7.6 6.5 1.6 57 10.2 7.6 2.8 56 9.2 7.6 55 10.1 7.8 2.0 5410.2 7.9 52 10.3 8.1 51 9.8 8.0 50 8.6 7.6 2.4 48 >9.0 8.2 66 >9.0 7.41.7 73 7.9 7.1 47 8.8 8.0 4.0 74 8.2 7.1 46 9.0 8.0 31 8.2 7.4 75 7.37.1 27 >9.0 8.0 45 9.0 6.7 1.7 44 8.2 7.4 1.8 43 8.5 7.8 2.1 29 7.9 6.942 8.3 7.5 71 7.5 6.5 70 8.5 6.8 69 7.6 6.7 25 7.6 7.0 41 8.4 5.8 40 8.57.5 23 8.4 7.5 22 9.1 7.2 26 >9.0 7.5 65 >9.0 7.5 1.6 39 9.0 7.5 2.0 309.0 7.5 2.1 38 >9.0 7.5 37 8.6 7.5 36 8.7 8.6 2.5 79 8.4 8.1 2.7 18 8.38.0 81 8.9 7.5

D. Composition Example: Film-Coated Tablets Preparation of Tablet Core

A mixture of 100 g of a compound of formula (I), 570 g lactose and 200 gstarch is mixed well and thereafter humidified with a solution of 5 gsodium dodecyl sulphate and 10 g polyvinyl-pyrrolidone in about 200 mlof water. The wet powder mixture is sieved, dried and sieved again. Thenthere is added 100 g microcrystalline cellulose and 15 g hydrogenatedvegetable oil. The whole is mixed well and compressed into tablets,giving 10.000 tablets, each comprising 10 mg of a compound of formula(I).

Coating

To a solution of 10 g methyl cellulose in 75 ml of denaturated ethanolthere is added a solution of 5 g of ethyl cellulose in 150 ml ofdichloromethane. Then there are added 75 ml of dichloromethane and 2.5ml 1,2,3-propanetriol 10 g of polyethylene glycol is molten anddissolved in 75 ml of dichloromethane. The latter solution is added tothe former and then there are added 2.5 g of magnesium octadecanoate, 5g of polyvinyl-pyrrolidone and 30 ml of concentrated colour suspensionand the whole is homogenated. The tablet cores are coated with the thusobtained mixture in a coating apparatus.

The invention claimed is:
 1. A method of inhibiting breast carcinoma ina subject comprising administering to the subject a therapeuticallyeffective amount of a compound of formula (I),

the N-oxide forms, the pharmaceutically acceptable addition salts andthe stereo-chemically isomeric forms thereof, wherein each n is aninteger with value 0, 1 or 2 and when n is 0 then a direct bond isintended; each m is an integer with value 1 or 2; each X isindependently N or CH; each Y is independently O, S, or NR⁴; whereineach R⁴ is hydrogen, C₁₋₆alkyl, C₁₋₆alkyloxyC₁₋₆alkyl, C₃₋₆cycloalkyl,C₃₋₆cycloalkylmethyl, phenylC₁₋₆alkyl, —C(═O)—CHR⁵R⁶ or —S(═O)₂—N(CH₃)₂;wherein each R⁵ and R⁶ is independently hydrogen, amino, C₁₋₆alkyl oraminoC₁₋₆alkyl; and when Y is NR⁴ and R² is on the 7-position of theindolyl then R² and R⁴ together can form the bivalent radical—(CH₂)₂—  (a-1), or—(CH₂)₃—  (a-2); R¹ is hydrogen, C₁₋₆alkyl, hydroxyC₁₋₆alkyl,C₁₋₆alkylsulfonyl, C₁₋₆alkylcarbonyl or mono- ordi(C₁₋₆alkyl)aminosulfonyl; R² is hydrogen, hydroxy, amino, halo,C₁₋₆alkyl, cyano, C₂₋₆alkenyl, polyhaloC₁₋₆alkyl, nitro, phenyl,C₁₋₆alkylcarbonyl, hydroxycarbonyl, C₁₋₆alkylcarbonylamino,C₁₋₆alkyloxy, or mono- or di(C₁₋₆alkyl)amino; R³ is hydrogen, C₁₋₆alkyl,or C₁₋₆alkyloxy; and when R² and R³ are on adjacent carbon atoms, theycan form the bivalent radical —O—CH₂—O—.
 2. The method of claim 1wherein the compound is: